Novel inositol polyphosphate kinase genes and uses thereof

ABSTRACT

This invention relates to newly identified polynucleotides and polypeptides in the phytic acid biosynthetic pathway, variants and derivatives of same; methods for making the polynucleotides, polypeptides, variants, derivatives and antagonists. In particular the invention relates to polynucleotides and polypeptides of the inositol polyphosphate kinase gene family. In particular this invention relates to using the newly identified polynucleotides and polypeptides to modulate the phytic acid biosynthesis in such a way as to decrease phytate and/or increase non-phytate phosphorous, especially in corn or soy animal feedstuffs.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Application SerialNo. 60/261,465 filed Jan. 12, 2001, which is herein incorporated byreference.

TECHNICAL FIELD

[0002] The present invention relates to the field of animal nutrition.Specifically, the present invention relates to the identification anduse of genes encoding enzymes involved in the metabolism of phytate inplants and the use of these genes and mutants thereof to reduce thelevels of phytate, and/or increase the levels of non-phytate phosphorusin food or feed.

BACKGROUND OF THE INVENTION

[0003] The role of phosphorous in animal nutrition is well recognized,it is a critical component of the skeleton, nucleic acids, cellmembranes and some vitamins. Though phosphorous is essential for thehealth of animals, not all phosphorous in feed is bioavailable.

[0004] Phytates are the major form of phosphorous in seeds, for examplephytate represents about 60-80% of total phosphorous in corn andsoybean. When seed-based diets are fed to non-ruminants, the consumedphytic acid forms salts with several important mineral nutrients, suchas potassium, calcium, and iron, and also binds proteins in theintestinal tract. These phytate complexes cannot be metabolized bymonogastric animals and are excreted, effectively acting asanti-nutritional factors by reducing the bioavailability of dietaryphosphorous and minerals. Phytate-bound phosphorous in animal excretaalso has a negative environmental impact, contributing to surface andground water pollution.

[0005] There have been two major approaches to reducing the negativenutritional and environmental impacts of phytate in seed. The firstinvolves post-harvest interventions, which increase the cost andprocessing time of feed. Post-harvest processing technologies removephytic acid by fermentation or by the addition of compounds, such asphytases.

[0006] The second is a genetic approach, which has been stronglycorrelated with undesirable agronomic characteristics. One geneticapproach involves developing crop germplasm with heritable reductions inseed phytic acid. Heritable quantitative variation in seed phytic acidhas been observed among lines in several crop species, but is alsohighly and positively correlated with less desirable characteristics.While some variability for phytic acid was observed, there was no changein non-phytate phosphorous, only 2% of the observed variation in phyticacid was heritable whereas 98% of the variation was attributed toenvironmental factors.

[0007] Another traditional genetic approach involves selecting lowphytate lines from a mutagenized population to produce germplasmseparated from the undesirable correlated traits seen in traditionalbreeding. Most mutant lines are a loss of function, presumably blockedin the phytic acid biosynthetic pathway, therefore low phytic acidaccumulation will likely be a recessive trait. In certain cases, thisapproach has revealed that homozygosity for substantially reducedphytate proved lethal.

[0008] A more modern genetic approach is transgenic technology, whichhas been used to increase phytase levels in plants. These transgenicplant tissues or seed have been used as dietary supplements, but thisapproach has not been used to reduce phytic acid accumulation in seed.

[0009] The biosynthetic route leading to phytate is complex and notcompletely understood. Without wishing to be bound by any particulartheory of the formation of phytate, it is believed that the synthesismay be mediated by a series of one or more ADP-phosphotransferases,ATP-dependent kinases and isomerases. A number of intermediates havebeen isolated including, for example, monophosphates such asD-myo-inositol 3-monophosphate, diphosphates (IP2s) such asD-myo-inositol 3,4-bisphosphate, triphosphates (IP3s) such asD-myo-inositol 3,4,6 trisphosphate, tetraphosphates (IP4s) such asD-myo-inositol 3,4,5,6-tetrakisphosphates, and pentaphosphates (IP5s)such as D-myo-inositol 1,3,4,5,6 pentakisphosphate. The phosphorylationof the IP5 to IP6 is found to be reversible. Several futile cycles ofdephosphorylation and rephosphorylation of the P5 and P6 forms have beenreported as well as a cycle involving glucose-6-phosphate→D-myo-inositol3-monophosphate-→myo-inositol; the last step being completelyreversible, indicating that control of metabolic flux through thispathway may be important

[0010] Based on the foregoing, there exists the need to improve thenutritional content of plants, particularly corn and soybean byincreasing non-phytate phosphorous and reducing seed phytate. Thisinvention differs from the foregoing approaches in that it providestools and reagents that allow the skilled artisan, by the applicationof, inter alia, transgenic methodologies to influence the metabolic fluxin respect to the phytic acid pathway.

SUMMARY OF THE INVENTION

[0011] Inositol polyphosphate kinases are a class of proteins originallydiscovered in yeast and identified as part of a signal transductionpathway. These enzymes can use several inositol phosphate species assubstrates with adenosine triphosphate (ATP) in a phosphorylationreaction yielding the products adenosine diphosphate (ADP) andphosphorylated inositol phosphate (n+1). This invention foresees usingthese nucleic acids or polypeptides, or variants thereof, to modulatethe flux through the phytic acid biosynthetic pathway in order toimprove the nutritional quality of feed, corn and soy in particular, andto reduce the environmental impact of animal waste by creating seed withhigher available phosphorous or lower phytate levels.

DETAILED DESCRIPTION OF THE INVENTION

[0012] DEFINITIONS

[0013] Units, prefixes, and symbols may be denoted in their SI acceptedform. Unless otherwise indicated, nucleic acids are written left toright in 5′ to 3′ orientation; amino acid sequences are written left toright in amino to carboxy orientation, respectively. Numeric rangesrecited within the specification are inclusive of the numbers definingthe range and include each integer within the defined range. Amino acidsmay be referred to herein by either their commonly known three lettersymbols or by the one-letter symbols recommended by the IUPAC-IUBBiochemical Nomenclature Commission. Nucleotides, likewise, may bereferred to by their commonly accepted single-letter codes. Unlessotherwise provided for, software, electrical, and electronics terms asused herein are as defined in The New IEEE Standard Dictionary ofElectrical and Electronics Terms (5th edition, 1993). The terms definedbelow are more fully defined by reference to the specification as awhole.

[0014] The term “isolated” refers to material, such as a nucleic acid ora protein, which is: (1) substantially or essentially free fromcomponents which normally accompany or interact with the material asfound in its naturally occurring environment or (2) if the material isin its natural environment, the material has been altered by deliberatehuman intervention to a composition and/or placed at a locus in the cellother than the locus native to the material.

[0015] As used herein, the term “nucleic acid” means a polynucleotideand includes single or multi-stranded polymers of deoxyribonucleotide orribonucleotide bases. Nucleic acids may also include fragments andmodified nucleotides. Therefore, as used herein, the terms “nucleicacid” and “polynucleotide” are used interchangably.

[0016] As used herein, “inositol polyphosphate kinase polynucleotide” isa nucleic acid of the present invention and means a nucleic acid, orfragment thereof, comprising a polynucleotide encoding a polypeptidewith inositol polyphosphate kinase activity or a useful fragmentthereof.

[0017] As used herein, “IPPK” means inositol polyphosphate kinase inregards to any nucleic acid or polypeptide of the present invention, orthe associated functional activity.

[0018] As used herein, “polypeptide” means proteins, protein fragments,modified proteins (e.g., glycosylated, phosphorylated, or othermodifications), amino acid sequences and synthetic amino acid sequences.The polypeptide can be modified or not. Therefore, as used herein,“polypeptide” and “protein” are used interchangably.

[0019] As used herein, “inositol polyphosphate kinase polypeptide” is apolypeptide of the present invention which is capable of phosphorylatingan appropriate inositol phosphate substrate and refers to one or moreamino acid sequences, in modified or unmodified form. The term is alsoinclusive of fragments, variants, homologs, alleles or precursors (e.g.,preproproteins or proproteins) or activity thereof.

[0020] As used herein, “plant” includes plants and plant parts includingbut not limited to plant cells and plant tissues such as leaves, stems,roots, flowers, pollen, and seeds.

[0021] As used herein, “promoter” includes reference to a region of DNAupstream from the start of transcription and involved in recognition andbinding of RNA polymerase and other proteins to initiate transcription.

[0022] By “fragment” is intended a portion of the nucleotide sequence ora portion of the amino acid sequence and hence protein encoded thereby.Fragments of a nucleotide sequence may encode protein fragments thatretain the biological activity of the native nucleic acid, functionalfragments. Alternatively, fragments of a nucleotide sequence that can beuseful as hybridization probes may not encode fragment proteinsretaining biological activity. Thus, fragments of a nucleotide sequenceare generally greater than 25, 50, 100,150, 200, 250, 300, 350, 400,450, 500, 600, or 700 nucleotides and up to and including the entirenucleotide sequence encoding the proteins of the invention. Generallythe probes are less than 1000 nucleotides and often less than 500nucleotides. Fragments of the invention include antisense sequences usedto decrease expression of the inventive polynucleotides. Such antisensefragments may vary in length ranging from greater than 25, 50, 100, 200,300, 400, 500, 600, or 700 nucleotides and up to and including theentire coding sequence.

[0023] By “functional equivalent” as applied to a polynucleotide or aprotein is intended a polynucleotide or a protein of sufficient lengthto modulate the level of IPPK protein activity in a plant cell. Apolynucleotide functional equivalent can be in sense or antisenseorientation.

[0024] By “variants” is intended substantially similar sequences.Generally, nucleic acid sequence variants of the invention will have atleast 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the nativenucleotide sequence, wherein the % sequence identity is based on theentire sequence and is determined by GAP 10 analysis using defaultparameters. Generally, polypeptide sequence variants of the inventionwill have at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 98%sequence identity to the native protein, wherein the % sequence identityis based on the entire sequence and is determined by GAP 10 analysisusing default parameters. GAP uses the algorithm of Needleman and Wunsch(J. Mol. Biol. 48:443-453,1970) to find the alignment of two completesequences that maximizes the number of matches and minimizes the numberof gaps.

[0025] As used herein “transformation” includes stable transformationand transient transformation.

[0026] As used herein “stable transformation” refers to the transfer ofa nucleic acid fragment into a genome of a host organism (this includesboth nuclear and organelle genomes) resulting in genetically stableinheritance. In addition to traditional methods, stable transformationincludes the alteration of gene expression by any means includingchimeraplasty or transposon insertion.

[0027] As used herein “transient transformation” refers to the transferof a nucleic acid fragment or protein into the nucleus (orDNA-containing organelle) of a host organism resulting in geneexpression without integration and stable inheritance.

[0028] “IPPK enzyme-binding molecule”, as used herein, refers tomolecules or ions which bind or interact specifically with phytatebiosynthetic enzyme polypeptides or polynucleotides of the presentinvention, including, for example enzyme substrates, cofactors,antagonists, inhibitors, cell membrane components and classicalreceptors. Binding between polypeptides of the invention and suchmolecules, including binding or interaction molecules may be exclusiveto polypeptides of the invention, or it may be highly specific forpolypeptides of the invention, or it may be highly specific to a groupof proteins that includes polypeptides of the invention, or it may bespecific to several groups of proteins at least one of which includes apolypeptide of the invention. Binding molecules also include antibodiesand antibody-derived reagents that bind specifically to polypeptides ofthe invention.

[0029] “High phosphorous transgenic”, as used herein, means an entitywhich, as a result of recombinant genetic manipulation, produces seedwith a heritable decrease in phytic acid percentage and/or increase innon-phytate phosphorous percentage as compared to a corresponding plantthat has not been transformed.

[0030] “Phytic acid”, as used herein, means myo-inositol tetraphosphoricacid, myo-inositol pentaphosphoric acid or myo-inositol hexaphosphoricacid. As a salt with cations, phytic acid is “phytate”.

[0031] “Non-phytate phosphorous”, as used herein, means total phosphorusminus phytate phosphorous.

[0032] “Non-ruminant animal” means an animal with a simple stomachdivided into the esophageal, cardia, fundus and pylorus regions. Anon-ruminant animal additionally implies a species of animal without afunctional rumen. A rumen is a section of the digestive system wherefeedstuff/food is soaked and subjected to digestion by microorganismsbefore passing on through the digestive tract. This phenomenon does notoccur in a non-ruminant animal. The term non-ruminant animal includesbut is not limited to humans, swine, poultry, cats and dogs.

NUCLEIC ACIDS

[0033] The inositol polyphosphate kinase gene family encodes a class ofenzymes capable of using several different inositol phosphates assubstrates in a phosphorylation reaction, using adenosine triphosphate(ATP) as the phosphate donor, resulting in the products adenosinediphosphate (ADP) and a phosphorylated inositol phosphate. It isexpected that modulating the expression and/or level of the nucleicacids of the present invention will modulate the phytate biosyntheticpathway providing methods to increase available phosphorous, decreasephytate and/or decrease polluting phytate-bound phosphorous in animalwaste.

[0034] The isolated nucleic acids of the present invention can be madeusing (a) standard recombinant methods, (b) synthetic techniques, orcombinations thereof. In some embodiments, the polynucleotides of thepresent invention can be cloned, amplified, or otherwise constructedfrom a monocot or dicot. Typical examples of monocots are corn, sorghum,barley, wheat, millet, rice, or turf grass. Typical dicots includesoybeans, safflower, sunflower, canola, alfalfa, potato, or cassava.

[0035] Functional fragments included in the invention can be obtainedusing primers which selectively hybridize under stringent conditions.Primers are generally at least 12 bases in length and can be as high as200 bases, but will generally be from 15 to 75, or more likely from 15to 50 bases. Functional fragments can be identified using a variety oftechniques such as restriction analysis, Southern analysis, primerextension analysis, and DNA sequence analysis.

[0036] The present invention includes a plurality of polynucleotidesthat encode for the identical amino acid sequence. The degeneracy of thegenetic code allows for such “silent variations” which can be used, forexample, to selectively hybridize and detect allelic variants ofpolynucleotides of the present invention. Additionally, the presentinvention includes isolated nucleic acids comprising allelic variants.The term “allele” as used herein refers to a related nucleic acid of thesame gene.

[0037] Variants of nucleic acids included in the invention can beobtained, for example, by oligonucleotide-directed mutagenesis,linker-scanning mutagenesis, mutagenesis using the polymerase chainreaction, and the like. See, for example, pages 8.0.3-8.5.9 CurrentProtocols in Molecular Biology, Ausubel et al., Eds., Greene Publishingand Wiley-lnterscience, New York (1995). Also, see generally, McPherson(ed.), DIRECTED MUTA GENESIS: A Practical Approach, (IRL Press, 1991).Thus, the present invention also encompasses DNA molecules comprisingnucleotide sequences that have substantial sequence similarity with theinventive sequences.

[0038] Variants included in the invention may contain individualsubstitutions, deletions or additions to the nucleic acid or polypeptidesequences which alter, add or delete a single amino acid or a smallpercentage of amino acids in the encoded sequence. A “conservativelymodified variant” is an alteration which results in the substitution ofan amino acid with a chemically similar amino acid. When the nucleicacid is prepared or altered synthetically, advantage can be taken ofknown codon preferences of the intended host.

[0039] The present invention also includes “shufflents” produced bysequence shuffling of the inventive polynucleotides to obtain a desiredcharacteristic. Sequence shuffling is described in PCT publication No.96/19256. See also, Zhang, J. H., et al., Proc. Natl. Acad. Sci. USA94:4504-4509 (1997).

[0040] The present invention also includes the use of 5′ and/or 3′ UTRregions for modulation of translation of heterologous coding sequences.Positive sequence motifs include translational initiation consensussequences (Kozak, Nucleic Acids Res.15:8125 (1987)) and the7-methylguanosine cap structure (Drummond et al., Nucleic Acids Res.13:7375 (1985)). Negative elements include stable intramolecular 5′ UTRstem-loop structures (Muesing et al., Cell 48:691 (1987)) and AUGsequences or short open reading frames preceded by an appropriate AUG inthe 5′ UTR (Kozak, supra, Rao et al., Mol. and Cell. Biol. 8:284(1988)).

[0041] Further, the polypeptide-encoding segments of the polynucleotidesof the present invention can be modified to alter codon usage. Alteredcodon usage can be employed to alter translational efficiency. Codonusage in the coding regions of the polynucleotides of the presentinvention can be analyzed statistically using commercially availablesoftware packages such as “Codon Preference” available from theUniversity of Wisconsin Genetics Computer Group (see Devereaux et al.,Nucleic Acids Res. 12:387-395 (1984)) or MacVector 4.1 (Eastman KodakCo., New Haven, Conn.).

[0042] For example, the inventive nucleic acids can be optimized forenhanced expression in plants of interest. See, for example, EPA0359472;WO91/16432; Perlak et al. (1991) Proc. Natl. Acad. Sci. USA88:3324-3328; and Murray et al. (1989) Nucleic Acids Res. 17:477-498. Inthis manner, the polynucleotides can be synthesized utilizingplant-preferred codons. See, for example, Murray et al. (1989) NucleicAcids Res. 17:477-498, the disclosure of which is incorporated herein byreference.

[0043] The present invention provides subsequences comprising isolatednucleic acids containing at least 20 contiguous bases of the inventivesequences. For example the isolated nucleic acid includes thosecomprising at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400,500, 600, 700 or 800 contiguous nucleotides of the inventive sequences.Subsequences of the isolated nucleic acid can be used to modulate ordetect gene expression by introducing into the subsequences compoundswhich bind, intercalate, cleave and/or crosslink to nucleic acids.

[0044] The nucleic acids of the invention may conveniently comprise amulti-cloning site comprising one or more endonuclease restriction sitesinserted into the nucleic acid to aid in isolation of thepolynucleotide. Also, translatable sequences may be inserted to aid inthe isolation of the translated polynucleotide of the present invention.For example, a hexa-histidine marker sequence provides a convenientmeans to purify the proteins of the present invention.

[0045] A polynucleotide of the present invention can be attached to avector, adapter, promoter, transit peptide or linker for cloning and/orexpression of a polynucleotide of the present invention. Additionalsequences may be added to such cloning and/or expression sequences tooptimize their function in cloning and/or expression, to aid inisolation of the polynucleotide, or to improve the introduction of thepolynucleotide into a cell. Use of cloning vectors, expression vectors,adapters, and linkers is well known and extensively described in theart. For a description of such nucleic acids see, for example,Stratagene Cloning Systems, Catalogs 1995, 1996, 1997 (La Jolla,Calif.); and, Amersham Life Sciences, Inc, Catalog '97 (ArlingtonHeights, Ill.).

[0046] The isolated nucleic acid compositions of this invention, such asRNA, cDNA, genomic DNA, or a hybrid thereof, can be obtained from plantbiological sources using any number of cloning methodologies known tothose of skill in the art. In some embodiments, oligonucleotide probeswhich selectively hybridize, under stringent conditions, to thepolynucleotides of the present invention are used to identify thedesired sequence in a cDNA or genomic DNA library.

[0047] Exemplary total RNA and mRNA isolation protocols are described inPlant Molecular Biology: A Laboratory Manual, Clark, Ed.,Springer-Verlag, Berlin (1997); and, Current Protocols in MolecularBiology, Ausubel et al., Eds., Greene Publishing and Wiley-Interscience,New York (1995). Total RNA and mRNA isolation kits are commerciallyavailable from vendors such as Stratagene (La Jolla, Calif.), Clonetech(Palo Alto, Calif.), Pharmacia (Piscataway, N.J.), and 5′-3′ (Paoli,Pa.). See also, U.S. Pat. Nos. 5,614,391; and, 5,459,253.

[0048] Typical cDNA synthesis protocols are well known to the skilledartisan and are described in such standard references as: PlantMolecular Biology: A Laboratory Manual, Clark, Ed., Springer-Verlag,Berlin (1997); and, Current Protocols in Molecular Biology, Ausubel etal., Eds., Greene Publishing and Wiley-Interscience, New York (1995).cDNA synthesis kits are available from a variety of commercial vendorssuch as Stratagene or Pharmacia.

[0049] An exemplary method of constructing a greater than 95% purefull-length cDNA library is described by Carninci et al., Genomics,37:327-336 (1996). Other methods for producing full-length libraries areknown in the art. See, e.g., Edery et al., Mol. Cell Biol.15(6):3363-3371 (1995); and PCT Application WO 96/34981.

[0050] It is often convenient to normalize a cDNA library to create alibrary in which each clone is more equally represented. A number ofapproaches to normalize cDNA libraries are known in the art.Construction of normalized libraries is described in Ko, Nucl. Acids.Res. 18(19):5705-5711 (1990); Patanjali et al., Proc. Natl. Acad. U.S.A.88:1943-1947 (1991); U.S. Pat. Nos. 5,482,685 and 5,637,685; and Soareset al., Proc. Natl. Acad. Sci. USA 91:9228-9232 (1994).

[0051] Subtracted cDNA libraries are another means to increase theproportion of less abundant cDNA species. See, Foote et al., in PlantMolecular Biology: A Laboratory Manual, Clark, Ed., Springer-Verlag,Berlin (1997); Kho and Zarbl, Technique 3(2):58-63 (1991); Sive and St.John, Nucl. Acids Res. 16(22):10937 (1988); Current Protocols inMolecular Biology, Ausubel et al., Eds., Greene Publishing andWiley-Interscience, New York (1995); and, Swaroop et al., Nucl. AcidsRes. 19(8):1954 (1991). cDNA subtraction kits are commerciallyavailable. See, e.g., PCR-Select (Clontech).

[0052] To construct genomic libraries, large segments of genomic DNA aregenerated by random fragmentation. Examples of appropriate molecularbiological techniques and instructions are found in Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring HarborLaboratory, Vols. 1-3 (1989), Methods in Enzymology, Vol. 152: Guide toMolecular Cloning Techniques, Berger and Kimmel, Eds., San Diego:Academic Press, Inc. (1987), Current Protocols in Molecular Biology,Ausubel et al., Eds., Greene Publishing and Wiley-lnterscience, New York(1995); Plant Molecular Biology: A Laboratory Manual, Clark, Ed.,Springer-Verlag, Berlin (1997). Kits for construction of genomiclibraries are also commercially available.

[0053] The cDNA or genomic library can be screened using a probe basedupon the sequence of a nucleic acid of the present invention such asthose disclosed herein. Probes may be used to hybridize with genomic DNAor cDNA sequences to isolate homologous polynucleotides in the same ordifferent plant species. Those of skill in the art will appreciate thatvarious degrees of stringency of hybridization can be employed in theassay; and either the hybridization or the wash medium can be stringent.The degree of stringency can be controlled by temperature, ionicstrength, pH and the presence of a partially denaturing solvent such asformamide.

[0054] Typically, stringent hybridization conditions will be those inwhich the salt concentration is less than about 1.5 M Na ion, typicallyabout 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to8.3 and the temperature is at least about 30° C. for short probes (e.g.,10 to 50 nucleotides) and at least about 60° C. for long probes (e.g.,greater than 50 nucleotides). Stringent conditions may also be achievedwith the addition of destabilizing agents such as formamide.

[0055] Exemplary low stringency conditions include hybridization with abuffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecylsulfate) at 37° C., and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 Mtrisodium citrate) at 50° C. Exemplary moderate stringency conditionsinclude hybridization in 40 to 45% formamide, 1 M NaCl, 1% SDS at 37°C., and a wash in 0.5× to 1×SSC at 55° C. Exemplary high stringencyconditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at37° C., and a wash in 0.1×SSC at 60° C. Typically the time ofhybridization is from 4 to 16 hours.

[0056] An extensive guide to the hybridization of nucleic acids is foundin Tijssen, Laboratory Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2“Overview of principles of hybridization and the strategy of nucleicacid probe assays”, Elsevier, New York (1993); and Current Protocols inMolecular Biology, Chapter 2, Ausubel et al., Eds., Greene Publishingand Wiley-Interscience, New York (1995). Often, cDNA libraries will benormalized to increase the representation of relatively rare cDNAs.

[0057] The nucleic acids of the invention can be amplified from nucleicacid samples using amplification techniques. For instance, polymerasechain reaction (PCR) technology can be used to amplify the sequences ofpolynucleotides of the present invention and related polynucleotidesdirectly from genomic DNA or cDNA libraries. PCR and other in vitroamplification methods may also be useful, for example, to clone nucleicacid sequences that code for proteins to be expressed, to make nucleicacids to use as probes for detecting the presence of the desired mRNA insamples, for nucleic acid sequencing, or for other purposes.

[0058] Examples of techniques useful for in vitro amplification methodsare found in Berger, Sambrook, and Ausubel, as well as Mullis et al.,U.S. Pat. No. 4,683,202 (1987); and, PCR Protocols A Guide to Methodsand Applications, Innis et al., Eds., Academic Press Inc., San Diego,Calif. (1990). Commercially available kits for genomic PCR amplificationare known in the art. See, e.g., Advantage-GC Genomic PCR Kit(Clontech). The T4 gene 32 protein (Boehringer Mannheim) can be used toimprove yield of long PCR products. PCR-based screening methods havealso been described. Wilfinger et al. describe a PCR-based method inwhich the longest cDNA is identified in the first step so thatincomplete clones can be eliminated from study. BioTechniques,22(3):481-486 (1997).

[0059] In one aspect of the invention, nucleic acids can be amplifiedfrom a plant nucleic acid library. The nucleic acid library may be acDNA library, a genomic library, or a library generally constructed fromnuclear transcripts at any stage of intron processing. Libraries can bemade from a variety of plant tissues such as ears, seedlings, leaves,stalks, roots, pollen, or seeds. Good results have been obtained usingtissues such as corn nucellus 5 days after silking, corn embryos 20 daysafter pollination, pooled primary and secondary immature ears from corn,corn leaves at the V3-V4 stage, 20 day old cold germinated cornseedlings, V5 corn roots, soybean 8 day old root tissue, eucalyptuscapsules (possibly fertile seed), and Guayule stem bark.

[0060] Alternatively, the sequences of the invention can be used toisolate corresponding sequences in other organisms, particularly otherplants, more particularly, other monocots. In this manner, methods suchas PCR, hybridization, and the like can be used to identify suchsequences having substantial sequence similarity to the sequences of theinvention. See, for example, Sambrook et al. (1989) Molecular Cloning: ALaboratory Manual (2d ed., Cold Spring Harbor Laboratory Press,Plainview, N.Y.). and Innis et al. (1990), PCR Protocols: A Guide toMethods and Applications (Academic Press, New York). Coding sequencesisolated based on their sequence identity to the entire inventive codingsequences set forth herein or to fragments thereof are encompassed bythe present invention.

[0061] The isolated nucleic acids of the present invention can also beprepared by direct chemical synthesis by methods such as thephosphotriester method of Narang et al., Meth. Enzymol. 68:90-99 (1979);the phosphodiester method of Brown et al., Meth. Enzymol. 68:109-151(1979); the diethylphosphoramidite method of Beaucage et al., Tetra.Lett. 22:1859-1862 (1981); the solid phase phosphoramidite triestermethod described by Beaucage and Caruthers, Tetra. Letts.22(20):1859-1862 (1981), e.g., using an automated synthesizer, e.g., asdescribed in Needham-VanDevanter et al., Nucleic Acids Res. 12:6159-6168(1984); and, the solid support method of U.S. Pat. No. 4,458,066.Chemical synthesis generally produces a single stranded oligonucleotide.This may be converted into double stranded DNA by hybridization with acomplementary sequence, or by polymerization with a DNA polymerase usingthe single strand as a template. One of skill will recognize that whilechemical synthesis of DNA is limited to sequences of about 100 bases,longer sequences may be obtained by the ligation of shorter sequences.

[0062] The nucleic acids of the present invention include thoseamplified using the following primer pairs: SEQ ID NOS: 26 and 27.

EXPRESSION CASSETTES

[0063] In another embodiment expression cassettes comprising isolatednucleic acids of the present invention are provided. An expressioncassette will typically comprise a polynucleotide of the presentinvention operably linked to transcriptional initiation regulatorysequences which will direct the transcription of the polynucleotide inthe intended host cell, such as tissues of a transformed plant.

[0064] The construction of such expression cassettes which can beemployed in conjunction with the present invention is well known tothose of skill in the art in light of the present disclosure. See, e.g.,Sambrook et al.; Molecular Cloning: A Laboratory Manual; Cold SpringHarbor, New York; (1989); Gelvin et al.; Plant Molecular Biology Manual(1990); Plant Biotechnology: Commercial Prospects and Problems, eds.Prakash et al.; Oxford & IBH Publishing Co.; New Delhi, India; (1993);and Heslot et al.; Molecular Biology and Genetic Engineering of Yeasts;CRC Press, Inc., USA; (1992); each incorporated herein in its entiretyby reference.

[0065] For example, plant expression vectors may include (1) a clonedplant gene under the transcriptional control of 5′ and 3′ regulatorysequences and (2) a dominant selectable marker. Such plant expressionvectors may also contain, if desired, a promoter regulatory region(e.g., one conferring inducible, constitutive, environmentally- ordevelopmentally-regulated, or cell- or tissue-specific/selectiveexpression), a transcription initiation start site, a ribosome bindingsite, an RNA processing signal, a transcription termination site, and/ora polyadenylation signal.

[0066] Constitutive, tissue-preferred or inducible promoters can beemployed. Examples of constitutive promoters include the cauliflowermosaic virus (CaMV) 35S transcription initiation region, the 1′- or 2′-promoter derived from T-DNA of Agrobacterium tumefaciens, the actinpromoter, the ubiquitin promoter, the histone H2B promoter (Nakayama etal., 1992, FEBS Lett 30:167-170), the Smas promoter, the cinnamylalcohol dehydrogenase promoter (U.S. Pat. No. 5,683,439), the Nospromoter, the pEmu promoter, the rubisco promoter, the GRP1-8 promoter,and other transcription initiation regions from various plant genesknown in the art.

[0067] Examples of inducible promoters are the Adh1 promoter which isinducible by hypoxia or cold stress, the Hsp70 promoter which isinducible by heat stress, the PPDK promoter which is inducible by light,the In2 promoter which is safener induced, the ERE promoter which isestrogen induced and the Pepcarboxylase promoter which is light induced.

[0068] Examples of promoters under developmental control includepromoters that initiate transcription preferentially in certain tissues,such as leaves, roots, fruit, pollen, seeds, or flowers. An exemplarypromoter is the anther specific promoter 5126 (U.S. Pat. Nos. 5,689,049and 5,689,051). Examples of seed-preferred promoters include, but arenot limited to, 27 kD gamma zein promoter and waxy promoter, (Boronat,A., et al., Plant Sci. 47:95-102 (1986); Reina, M., et al., NucleicAcids Res. 18(21):6426 (1990); Kloesgen, R. B., et al., Mol. Gen. Genet.203:237-244 (1986)), as well as the globulin 1, oleosin and thephaseolin promoters. The disclosures each of these are incorporatedherein by reference in their entirety.

[0069] The barley or maize Nuc1 promoter, the maize Cim1 promoter or themaize LTP2 promoter can be used to preferentially express in thenucellus. See, for example WO 00/11177, the disclosure of which isincorporated herein by reference.

[0070] Either heterologous or non-heterologous (i.e., endogenous)promoters can be employed to direct expression of the nucleic acids ofthe present invention. These promoters can also be used, for example, inexpression cassettes to drive expression of antisense nucleic acids toreduce, increase, or alter concentration and/or composition of theproteins of the present invention in a desired tissue.

[0071] If polypeptide expression is desired, it is generally desirableto include a polyadenylation region at the 3′-end of a polynucleotidecoding region. The polyadenylation region can be derived from thenatural gene, from a variety of other plant genes, or from T-DNA. The 3′end sequence to be added can be derived from, for example, the nopalinesynthase or octopine synthase genes, or alternatively from another plantgene, or less preferably from any other eukaryotic gene.

[0072] An intron sequence can be added to the 5′ untranslated region orthe coding sequence of the partial coding sequence to increase theamount of the mature message that accumulates. See for example Buchmanand Berg, Mol. Cell Biol. 8:4395-4405 (1988); Callis et al., Genes Dev.1:1183-1200 (1987). Use of maize introns Adh1-S intron 1, 2, and 6, theBronze-1 intron are known in the art. See generally, The Maize Handbook,Chapter 116, Freeling and Walbot, Eds., Springer, N.Y. (1994).

[0073] The vector comprising the sequences from a polynucleotide of thepresent invention will typically comprise a marker gene which confers aselectable phenotype on plant cells. Usually, the selectable marker geneencodes antibiotic or herbicide resistance. Suitable genes include thosecoding for resistance to the antibiotics spectinomycin and streptomycin(e.g., the aada gene), the streptomycin phosphotransferase (SPT) genecoding for streptomycin resistance, the neomycin phosphotransferase(NPTII) gene encoding kanamycin or geneticin resistance, the hygromycinphosphotransferase (HPT) gene coding for hygromycin resistance.

[0074] Suitable genes coding for resistance to herbicides include thosewhich act to inhibit the action of acetolactate synthase (ALS), inparticular the sulfonylurea-type herbicides (e.g., the acetolactatesynthase (ALS) gene containing mutations leading to such resistance inparticular the S4 and/or Hra mutations), those which act to inhibitaction of glutamine synthase, such as phosphinothricin or basta (e.g.,the bar gene), or other such genes known in the art. The bar geneencodes resistance to the herbicide basta and the ALS gene encodesresistance to the herbicide chlorsulfuron.

[0075] Typical vectors useful for expression of genes in higher plantsare well known in the art and include vectors derived from thetumor-inducing (Ti) plasmid of Agrobacterium tumefaciens described byRogers et al., Meth. In Enzymol. 153:253-277 (1987). Exemplary A.tumefaciens vectors useful herein are plasmids pKYLX6 and pKYLX7 ofSchardl et al., Gene 61:1-11 (1987) and Berger et al., Proc. Natl. Acad.Sci. USA 86:8402-8406 (1989). Another useful vector herein is plasmidpBI101.2 that is available from Clontech Laboratories, Inc. (Palo Alto,Calif.).

[0076] A variety of plant viruses that can be employed as vectors areknown in the art and include cauliflower mosaic virus (CaMV),geminivirus, brome mosaic virus, and tobacco mosaic virus.

[0077] A polynucleotide of the present invention can be expressed ineither sense or anti-sense orientation as desired. In plant cells, ithas been shown that antisense RNA inhibits gene expression by preventingthe accumulation of mRNA which encodes the enzyme of interest, see,e.g., Sheehy et al., Proc. Natl. Acad. Sci. USA 85:8805-8809 (1988); andHiatt et al., U.S. Pat. No. 4,801,340.

[0078] Another method of suppression is sense suppression. Introductionof nucleic acid configured in the sense orientation has been shown to bean effective means by which to block the transcription of target genes.For an example of the use of this method to modulate expression ofendogenous genes see, Napoli et al., The Plant Cell 2:279-289 (1990) andU.S. Pat. No. 5,034,323.

[0079] Recent work has shown suppression with the use of double strandedRNA. Such work is described in Tabara et al., Science 282:5388:430431(1998), WO 99/53050 and WO 98/53083.

[0080] Catalytic RNA molecules or ribozymes can also be used to inhibitexpression of plant genes. The inclusion of ribozyme sequences withinantisense RNAs confers RNA-cleaving activity upon them, therebyincreasing the activity of the constructs. The design and use of targetRNA-specific ribozymes is described in Haseloff et al., Nature334:585-591 (1988).

[0081] A variety of cross-linking agents, alkylating agents and radicalgenerating species as pendant groups on polynucleotides of the presentinvention can be used to bind, label, detect, and/or cleave nucleicacids. For example, Vlassov, V. V., et al., Nucleic Acids Res (1986)14:4065-4076, describe covalent bonding of a single-stranded DNAfragment with alkylating derivatives of nucleotides complementary totarget sequences. A report of similar work by the same group is that byKnorre, D. G., et al., Biochimie (1985) 67:785-789. Iverson and Dervanalso showed sequence-specific cleavage of single-stranded DNA mediatedby incorporation of a modified nucleotide which was capable ofactivating cleavage (J. Am. Chem. Soc. (1987) 109:1241-1243). Meyer, R.B., et al., J. Am. Chem. Soc. (1989) 111:8517-8519, effect covalentcrosslinking to a target nucleotide using an alkylating agentcomplementary to the single-stranded target nucleotide sequence. Aphotoactivated crosslinking to single-stranded oligonucleotides mediatedby psoralen was disclosed by Lee, B. L., et al., Biochemistry (1988)27:3197-3203. Use of crosslinking in triple-helix forming probes wasalso disclosed by Home et al., J. Am. Chem. Soc. (1990) 112:2435-2437.Use of N4, N4-ethanocytosine as an alkylating agent to crosslink tosingle-stranded oligonucleotides has also been described by Webb andMatteucci, J. Am. Chem. Soc. (1986) 108:2764-2765; Nucleic Acids Res(1986) 14:7661-7674; Feteritz et al., J. Am. Chem. Soc. 113:4000 (1991).Various compounds to bind, detect, label, and/or cleave nucleic acidsare known in the art. See, for example, U.S. Pat. Nos. 5,543,507;5,672,593; 5,484,908; 5,256,648; and, 5,681941.

GENE OR TRAIT STACKING

[0082] In certain embodiments the nucleic acid sequences of the presentinvention can be stacked with any combination of polynucleotidesequences of interest in order to create plants with a desiredphenotype. For example, the polynucleotides of the present invention maybe stacked with any other polynucleotides of the present invention, suchas any combination of IPPKs (SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13, 15, 20,22, and 24), or with other genes implicated in phytic acid metabolicpathways such as phytase; Lpa1, Lpa2 (see U.S. Pat. Nos. 5,689,054 and6,111,168); myo-inositol 1-phosphate synthase (MI1PS), inositol1,3,4-trisphosphate ⅚ kinases (ITPKs) and myo-inositol monophophatase(IMP) (see U.S. Provisional Application Serial No. 60/325,308 filed Sep.27, 2001, and WO 99/05298) and the like, the disclosures of which areherein incorporated by reference. The combinations generated can alsoinclude multiple copies of any one of the polynucleotides of interest.The polynucleotides of the present invention can also be stacked withany other gene or combination of genes to produce plants with a varietyof desired trait combinations including but not limited to traitsdesirable for animal feed such as high oil genes (e.g., U.S. Pat. No.6,232,529); balanced amino acids (e.g. hordothionins (U.S. Pat. Nos.5,990,389; 5,885,801; 5,885,802; and 5,703,409); barley high lysine(Williamson et al. (1987) Eur. J. Biochem. 165:99-106; and WO 98/20122);and high methionine proteins (Pedersen et al. (1986) J. Biol. Chem.261:6279; Kirihara et al. (1988) Gene 71:359; and Musumura et al. (1989)Plant Mol. Biol. 12: 123)); increased digestibility (e.g., modifiedstorage proteins (U.S. Provisional Application Serial No. 60,246,455,filed Nov. 11, 2000); and thioredoxins (U.S. Provisional ApplicationSerial No. 60/250,705, filed Dec. 12, 2000)), the disclosures of whichare herein incorporated by reference. The polynucleotides of the presentinvention can also be stacked with traits desirable for insect, diseaseor herbicide resistance (e.g. Bacillus thuringiensis toxic proteins(U.S. Pat. Nos. 5,366,892; 5,747,450; 5,737,514; 5723,756; 5,593,881;Geiser et al (1986) Gene 48:109); lectins (Van Damme et al. (1994) PlantMol. Biol. 24:825); fumonisin detoxification genes (U.S. Pat. No.5,792,931); avirulence and disease resistance genes (Jones et al. (1994)Science 266:789; Martin et al. (1993) Science 262:1432; Mindrinos et al.(1994) Cell 78:1089); acetolactate synthase (ALS) mutants that lead toherbicide resistance such as the S4 and/or Hra mutations; inhibitors ofglutamine synthase such as phosphinothricin or basta (e.g., bar gene);and glyphosate resistance (EPSPS gene)); and traits desirable forprocessing or process products such as high oil (e.g., U.S. Pat. No.6,232,529 ); modified oils (e.g., fatty acid desaturase genes (U.S. Pat.No. 5,952,544; WO 94/11516)); modified starches (e.g., ADPGpyrophosphorylases (AGPase), starch synthases (SS), starch branchingenzymes (SBE) and starch debranching enzymes (SDBE)); and polymers orbioplastics (e.g., U.S. Pat. No. 5.602,321; beta-ketothiolase,polyhydroxybutyrate synthase, and acetoacetyl-CoA reductase (Schubert etal. (1988) J. Bacteriol. 170:5837-5847) facilitate expression ofpolyhydroxyalkanoates (PHAs)), the disclosures of which are hereinincorporated by reference. One could also combine the polynucleotides ofthe present invention with polynucleotides providing agronomic traitssuch as male sterility (e.g., see U.S. Pat. No. 5.583,210), stalkstrength, flowering time, or transformation technology traits such ascell cycle regulation or gene targeting (e.g. WO 99/61619; WO 00/17364;WO 99/25821), the disclosures of which are herein incorporated byreference. These stacked combinations can be created by any methodincluding but not limited to cross breeding plants by any conventionalor TopCross methodology, or genetic transformation. If the traits arestacked by genetically transforming the plants, the polynucleotidesequences of interest can be combined at any time and in any order. Forexample, a transgenic plant comprising one or more desired traits can beused as the target to introduce further traits by subsequenttransformation. The traits can be introduced simultaneously in aco-transformation protocol with the polynucleotides of interest providedby any combination of transformation cassettes. For example, if twosequences will be introduced, the two sequences can be contained inseparate transformation cassettes (trans) or contained on the sametransformation cassette (cis). Expression of the sequences can be drivenby the same promoter or by different promoters. In certain cases, it maybe desirable to introduce a transformation cassette that will suppressthe expression of the polynucleotide of interest. This may be combinewith any combination of other suppression cassettes or overexpressioncassettes to generate the desired combination of traits in the plant.

PROTEINS

[0083] IPPK proteins are a class of proteins in inositol phosphatemetabolism that are all involved in the phosphorylation of theirappropriate inositol phosphate substrates, including but not limited toIP2, IP3, IP4, and IP5, using ATP as the phosphate donor. The sequencesof the present invention have homology to a conserved inositol phosphatebinding motif domain show in SEQ ID NO: 29. Analysis of the polypeptidesequences of the present invention reveals the consensus domains shownin SEQ ID NOS: 30-37. It is expected that modulation of the expressionof these proteins of the present invention will provide methods toimprove the quality of animal feed by reducing the level of phytateand/or increasing the level of bioavailable phosphorous. Reducingphytate levels should also result in less environment-pollutingphosphorous in the waste of non-ruminant animals.

[0084] Proteins of the present invention include proteins having thedisclosed sequences as well proteins coded by the disclosedpolynucleotides. In addition, proteins of the present invention includeproteins derived from the native protein by deletion, addition orsubstitution of one or more amino acids at one or more sites in thenative protein. Such variants may result from, for example, geneticpolymorphism or from human manipulation. Methods for such manipulationsare generally known in the art.

[0085] For example, amino acid sequence variants of the polypeptide canbe prepared by mutations in the cloned DNA sequence encoding the nativeprotein of interest. Methods for mutagenesis and nucleotide sequencealterations are well known in the art. See, for example, Walker andGaastra, eds. (1983) Techniques in Molecular Biology (MacMillanPublishing Company, New York); Kunkel (1985) Proc. Natl. Acad. Sci. USA82:488-492; Kunkel et al. (1987) Methods Enzymol. 154:367-382; Sambrooket al. (1989) Molecular Cloning: A Laboratory Manual (Cold SpringHarbor, N.Y.); U.S. Pat. No. 4,873,192; and the references citedtherein; herein incorporated by reference. Guidance as to appropriateamino acid substitutions that do not affect biological activity of theprotein of interest may be found in the model of Dayhoff et al. (1978)Atlas of Protein Sequence and Structure (Natl. Biomed. Res. Found.,Washington, D.C.), herein incorporated by reference. Conservativesubstitutions, such as exchanging one amino acid with another havingsimilar properties, may be preferred.

[0086] In constructing variants of the proteins of interest,modifications to the nucleotide sequences encoding the variants cangenerally be made such that variants continue to possess the desiredactivity.

[0087] The isolated proteins of the present invention include apolypeptide comprising at least 25 contiguous amino acids encoded by anyone of the nucleic acids of the present invention, or polypeptides thatare conservatively modified variants thereof. The proteins of thepresent invention or variants thereof can comprise any number ofcontiguous amino acid residues from a polypeptide of the presentinvention, wherein that number is selected from the group of integersconsisting of from 25 to the number of residues in a full-lengthpolypeptide of the present invention. Optionally, this subsequence ofcontiguous amino acids is at least 25, 30, 40, 50, 60, 70, 80, 90,100,150, 200, 250, 300, 350, 400, 450, or 500 amino acids in length.

[0088] The present invention includes catalytically active polypeptides(i.e., enzymes). Catalytically active polypeptides will generally have aspecific activity of at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%,90%, or 95% that of the native (non-synthetic), endogenous polypeptide.Further, the substrate specificity (k_(cat)/K_(m)) is optionallysubstantially similar to the native (non-synthetic), endogenouspolypeptide. Typically, the K_(m) will be at least about 30%, 40%, 50%,60%, 70%, 80%, 90%, or 95% that of the native (non-synthetic),endogenous polypeptide. Methods of assaying and quantifying measures ofenzymatic activity and substrate specificity (k_(cat)/K_(m)), are wellknown to those of skill in the art. See, e.g., Segel, BiochemicalCalculations, 2^(nd) ed., John Wiley and Sons, New York (1976).

[0089] The present invention includes modifications that can be made toan inventive protein. In particular, it may be desirable to diminish theactivity of the gene. Other modifications may be made to facilitate thecloning, expression, or incorporation of the targeting molecule into afusion protein. Such modifications are well known to those of skill inthe art and include, for example, a methionine added at the aminoterminus to provide an initiation site, or additional amino acids (e.g.,poly His) placed on either terminus to create conveniently locatedrestriction sites or termination codons or purification sequences.

[0090] Using the nucleic acids of the present invention, one may expressa protein of the present invention in recombinantly engineered cellssuch as bacteria, yeast, insect, mammalian, or plant cells. The cellsproduce the protein in a non-natural condition (e.g., in quantity,composition, location, and/or time), because they have been geneticallyaltered through human intervention to do so.

[0091] Typically, an intermediate host cell may be used in the practiceof this invention to increase the copy number of the cloning vector.With an increased copy number, the vector containing the gene ofinterest can be isolated in significant quantities for introduction intothe desired plant cells.

[0092] Host cells that can be used in the practice of this inventioninclude prokaryotes and eukaryotes. Prokaryotes include bacterial hostssuch as Eschericia coli, Salmonella typhimurium, and Serratiamarcescens. Eukaryotic hosts such as yeast, insect cells or filamentousfungi may also be used in this invention.

[0093] Commonly used prokaryotic control sequences include such commonlyused promoters as the beta lactamase (penicillinase) and lactose (lac)promoter systems (Chang et al., Nature 198:1056 (1977)), the tryptophan(trp) promoter system (Goeddel et al., Nucleic Acids Res. 8:4057 (1980))and the lambda derived P L promoter and N-gene ribosome binding site(Shimatake et al., Nature 292:128 (1981)). The inclusion of selectionmarkers in DNA vectors transfected in E. coli is also useful. Examplesof such markers include genes specifying resistance to ampicillin,tetracycline, or chloramphenicol.

[0094] The vector is selected to allow introduction into the appropriatehost cell. Bacterial vectors are typically of plasmid or phage origin.Expression systems for expressing a protein of the present invention areavailable using Bacillus sp. and Salmonella (Palva et al., Gene22:229-235 (1983); Mosbach et al., Nature 302:543-545 (1983)).

[0095] Synthesis of heterologous proteins in yeast is well known. SeeSherman, F., et al., Methods in Yeast Genetics, Cold Spring HarborLaboratory (1982). Two widely utilized yeast for production ofeukaryotic proteins are Saccharomyces cerevisiae and Pichia pastoris.Vectors, strains, and protocols for expression in Saccharomyces andPichia are known in the art and available from commercial suppliers(e.g., Invitrogen). Suitable vectors usually have expression controlsequences, such as promoters, including 3-phosphoglycerate kinase oralcohol oxidase, and an origin of replication, termination sequences andthe like as desired.

[0096] The baculovirus expression system (BES) is a eukaryotic,helper-independent expression system which has been used to expresshundreds of foreign genes (Luckow, V., Ch. 4 “Cloning and Expression ofHeterologous Genes in Insect Cells with Baculovirus Vectors” inRecombinant DNA Technology and Applications, A. Prokopet al., Eds.McGraw-Hill, Inc. (1991); Luckow, V., Ch. 10 “Insect ExpressionTechnology” in Principles & Practice of Protein Engineering, J. L.Cleland and C. S. Craig, Eds. John Wiley & Sons, (1994)).

[0097] Recombinant baculoviruses are generated by inserting theparticular gene- or genes-of-interest into the baculovirus genome usingestablished protocols with vectors and reagents from commercialsuppliers (e.g., Invitrogen, Life Technologies Incorporated). Commercialvectors are readily available with various promoters, such as polyhedrinand p10, optional signal sequences for protein secretion, or affinitytags, such as 6×histidine. These recombinant viruses are grown,maintained and propagated in commercially available cell lines derivedfrom several insect species including Spodoptera frugiperda andTrichoplusia ni. The insect cells can be cultured using well-establishedprotocols in a variety of different media, for example, with and withoutbovine serum supplementation. The cultured cells are infected with therecombinant viruses and the gene-of-interest polypeptide is expressed.Proteins expressed with the baculovirus system have been extensivelycharacterized and, in many cases, their post-translational modificationssuch as phosphorylation, acylation, etc., are identical to the nativelyexpressed protein.

[0098] A protein of the present invention, once expressed, can beisolated from cells by lysing the cells and applying standard proteinisolation techniques to the lysates. The monitoring of the purificationprocess can be accomplished by using Western blot techniques orradioimmunoassay or other standard immunoassay techniques. Expressioncassettes are also available which direct the expressed protein to besecreted from the cell into the media. In these cases, the expressedprotein can be purified from the cell growth media using standardprotein purification techniques.

[0099] The proteins of the present invention can also be constructedusing non-cellular synthetic methods. Solid phase synthesis of proteinsof less than about 50 amino acids in length may be accomplished byattaching the C-terminal amino acid of the sequence to an insolublesupport followed by sequential addition of the remaining amino acids inthe sequence. Techniques for solid phase synthesis are described byBarany and Merrifield, Solid-Phase Peptide Synthesis, pp. 3-284 in ThePeptides: Analysis, Synthesis, Biology. Vol. 2: Special Methods inPeptide Synthesis, Part A.; Merrifield et al., J. Am. Chem. Soc.85:2149-2156 (1963), and Stewart et al., Solid Phase Peptide Synthesis,2nd ed., Pierce Chem. Co., Rockford, Ill. (1984). Proteins of greaterlength may be synthesized by condensation of the amino and carboxytermini of shorter fragments. Methods of forming peptide bonds byactivation of a carboxy terminal end (e.g., by the use of the couplingreagent N,N′-dicyclohexylcarbodiimide)) are known to those of skill.

[0100] The proteins of this invention, recombinant or synthetic, may bepurified to substantial purity by standard techniques well known in theart, including detergent solubilization, selective precipitation withsuch substances as ammonium sulfate, column chromatography,immunopurification methods, and others. See, for instance, R. Scopes,Protein Purification: Principles and Practice, Springer-Verlag: New York(1982); Deutscher, Guide to Protein Purification, Academic Press (1990).For example, antibodies may be raised to the proteins as describedherein. Purification from E. coli can be achieved following proceduresdescribed in U.S. Pat. No. 4,511,503. Detection of the expressed proteinis achieved by methods known in the art and include, for example,radioimmunoassays, Western blotting techniques or immunoprecipitation.

[0101] The present invention further provides a method for modulating(i.e., increasing or decreasing) the concentration or composition of thepolypeptides of the present invention in a plant or part thereof.Modulation can be effected by increasing or decreasing the concentrationand/or the composition (i.e., the ratio of the polypeptides of thepresent invention) in a plant.

[0102] The method comprises transforming a plant cell with an expressioncassette comprising a polynucleotide of the present invention to obtaina transformed plant cell, growing the transformed plant cell underconditions allowing expression of the polynucleotide in the plant cellin an amount sufficient to modulate concentration and/or composition inthe plant cell.

[0103] In some embodiments, the content and/or composition ofpolypeptides of the present invention in a plant may be modulated byaltering, in vivo or in vitro, the promoter of a non-isolated gene ofthe present invention to up- or down-regulate gene expression. In someembodiments, the coding regions of native genes of the present inventioncan be altered via substitution, addition, insertion, or deletion todecrease activity of the encoded enzyme. See, e.g., Kmiec, U.S. Pat. No.5,565,350; Zarling et al., PCT/US93/03868. One method of down-regulationof the protein involves using PEST sequences that provide a target fordegradation of the protein.

[0104] In some embodiments, an isolated nucleic acid (e.g., a vector)comprising a promoter sequence is transfected into a plant cell.Subsequently, a plant cell comprising the promoter operably linked to apolynucleotide of the present invention is selected for by means knownto those of skill in the art such as, but not limited to, Southern blot,DNA sequencing, or PCR analysis using primers specific to the promoterand to the gene and detecting amplicons produced therefrom. A plant orplant part altered or modified by the foregoing embodiments is grownunder plant forming conditions for a time sufficient to modulate theconcentration and/or composition of polypeptides of the presentinvention in the plant. Plant forming conditions are well known in theart.

[0105] In general, content of the polypeptide is increased or decreasedby at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% relativeto a native control plant, plant part, or cell lacking theaforementioned expression cassette. Modulation in the present inventionmay occur during and/or subsequent to growth of the plant to the desiredstage of development. Modulating nucleic acid expression temporallyand/or in particular tissues can be controlled by employing theappropriate promoter operably linked to a polynucleotide of the presentinvention in, for example, sense or antisense orientation as discussedin greater detail, supra. Induction of expression of a polynucleotide ofthe present invention can also be controlled by exogenous administrationof an effective amount of inducing compound. Inducible promoters andinducing compounds which activate expression from these promoters arewell known in the art. In certain embodiments, the polypeptides of thepresent invention are modulated in monocots or dicots, for examplemaize, soybeans, sunflower, safflower, sorghum, canola, wheat, alfalfa,rice, barley and millet.

[0106] Means of detecting the proteins of the present invention are notcritical aspects of the present invention. The proteins can be detectedand/or quantified using any of a number of well-recognized immunologicalbinding assays (see, e.g., U.S. Pat. Nos. 4,366,241; 4,376,110;4,517,288; and 4,837,168). Fora review of the general immunoassays, seealso Methods in Cell Biology, Vol. 37: Antibodies in Cell Biology, Asai,Ed., Academic Press, Inc. New York (1993); Basic and Clinical Immunology7th Edition, Stites & Terr, Eds. (1991). Moreover, the immunoassays ofthe present invention can be performed in any of several configurations,e.g., those reviewed in Enzyme Immunoassay, Maggio, Ed., CRC Press, BocaRaton, Fla. (1980); Tijan, Practice and Theory of Enzyme Immunoassays,Laboratory Techniques in Biochemistry and Molecular Biology, ElsevierScience Publishers B. V., Amsterdam (1985); Harlow and Lane, supra;Immunoassay: A Practical Guide, Chan, Ed., Academic Press, Orlando, Fla.(1987); Principles and Practice of Immunoassays, Price and Newman Eds.,Stockton Press, NY (1991); and Non-isotopic Immunoassays, Ngo, Ed.,Plenum Press, NY (1988).

[0107] Typical methods include Western blot (immunoblot) analysis,analytic biochemical methods such as electrophoresis, capillaryelectrophoresis, high performance liquid chromatography (HPLC), thinlayer chromatography (TLC), hyperdiffusion chromatography, and the like,and various immunological methods such as fluid or gel precipitinreactions, immunodiffusion (single or double), immunoelectrophoresis,radioimmunoassays (RIAs), enzyme-linked immunosorbent assays (ELISAs),immunofluorescent assays, and the like.

[0108] Non-radioactive labels are often attached by indirect means.Generally, a ligand molecule (e.g., biotin) is covalently bound to themolecule. The ligand then binds to an anti-ligand (e.g., streptavidin)molecule which is either inherently detectable or covalently bound to asignal system, such as a detectable enzyme, a fluorescent compound, or achemiluminescent compound. A number of ligands and anti-ligands can beused. Where a ligand has a natural anti-ligand, for example, biotin,thyroxine, and cortisol, it can be used in conjunction with the labeled,naturally occurring anti-ligands. Alternatively, any haptenic orantigenic compound can be used in combination with an antibody.

[0109] The molecules can also be conjugated directly to signalgenerating compounds, e.g., by conjugation with an enzyme orfluorophore. Enzymes of interest as labels will primarily be hydrolases,particularly phosphatases, esterases and glycosidases, oroxidoreductases, particularly peroxidases. Fluorescent compounds includefluorescein and its derivatives, rhodamine and its derivatives, dansyl,umbelliferone, etc. Chemiluminescent compounds include luciferin, and2,3-dihydrophthalazinediones, e.g., luminol. For a review of variouslabeling or signal producing systems which may be used, see, U.S. Pat.No. 4,391,904, which is incorporated herein by reference.

[0110] Some assay formats do not require the use of labeled components.For instance, agglutination assays can be used to detect the presence ofthe target antibodies. In this case, antigen-coated particles areagglutinated by samples comprising the target antibodies. In thisformat, none of the components need be labeled and the presence of thetarget antibody is detected by simple visual inspection.

[0111] The proteins of the present invention can be used for identifyingcompounds that bind to (e.g., substrates), and/or increase or decrease(i.e., modulate) the enzymatic activity of catalytically activepolypeptides of the present invention. The method comprises contacting apolypeptide of the present invention with a compound whose ability tobind to or modulate enzyme activity is to be determined. The polypeptideemployed will have at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or95% of the specific activity of the native, full-length polypeptide ofthe present invention (e.g., enzyme). Methods of measuring enzymekinetics are well known in the art. See, e.g., Segel, BiochemicalCalculations, 2^(nd) ed., John Wiley and Sons, New York (1976).

[0112] Antibodies can be raised to a protein of the present invention,including individual, allelic, strain, or species variants, andfragments thereof, both in their naturally occurring (full-length) formsand in recombinant forms. Additionally, antibodies are raised to theseproteins in either their native configurations or in non-native nativeconfigurations. Anti-idiotypic antibodies can also be generated. Manymethods of making antibodies are known to persons of skill.

[0113] In some instances, it is desirable to prepare monoclonalantibodies from various mammalian hosts, such as mice, rodents,primates, humans, etc. Description of techniques for preparing suchmonoclonal antibodies are found in, e.g., Basic and Clinical Immunology,4th ed., Stites et al., Eds., Lange Medical Publications, Los Altos,Calif., and references cited therein; Harlow and Lane, Supra; Goding,Monoclonal Antibodies: Principles and Practice, 2nd ed., Academic Press,New York, N.Y. (1986); and Kohler and Milstein, Nature 256:495-497(1975).

[0114] Other suitable techniques involve selection of libraries ofrecombinant antibodies in phage or similar vectors (see, e.g., Huse etal., Science 246:1275-1281 (1989); and Ward et al., Nature 341:544-546(1989); and Vaughan et al., Nature Biotechnology 14:309-314 (1996)).Alternatively, high avidity human monoclonal antibodies can be obtainedfrom transgenic mice comprising fragments of the unrearranged humanheavy and light chain Ig loci (i.e., minilocus transgenic mice).Fishwild et al., Nature Biotech. 14:845-851 (1996). Also, recombinantimmunoglobulins may be produced. See, Cabilly, U.S. Pat. No. 4,816,567;and Queen et al., Proc. Natl. Acad. Sci. U.S.A. 86:10029-10033 (1989).

[0115] The antibodies of this invention can be used for affinitychromatography in isolating proteins of the present invention, forscreening expression libraries for particular expression products suchas normal or abnormal protein or for raising anti-idiotypic antibodieswhich are useful for detecting or diagnosing various pathologicalconditions related to the presence of the respective antigens.

[0116] Frequently, the proteins and antibodies of the present inventionmay be labeled by joining, either covalently or non-covalently, asubstance which provides for a detectable signal. A wide variety oflabels and conjugation techniques are known and are reported extensivelyin both the scientific and patent literature. Suitable labels includeradionucleotides, enzymes, substrates, cofactors, inhibitors,fluorescent moieties, chemiluminescent moieties, magnetic particles, andthe like.

TRANSFORMATION OF CELLS

[0117] The method of transformation is not critical to the presentinvention; various methods of transformation are currently available. Asnewer methods are available to transform crops or other host cells theymay be directly applied. Accordingly, a wide variety of methods havebeen developed to insert a DNA sequence into the genome of a host cellto obtain the transcription and/or translation of the sequence to effectphenotypic changes in the organism. Thus, any method which provides forefficient transformation/transfection may be employed.

[0118] A DNA sequence coding for the desired polynucleotide of thepresent invention, for example a cDNA or a genomic sequence encoding afull length protein, can be used to construct an expression cassettewhich can be introduced into the desired plant. Isolated nucleic acidacids of the present invention can be introduced into plants accordingto techniques known in the art. Generally, expression cassettes asdescribed above and suitable for transformation of plant cells areprepared.

[0119] Techniques for transforming a wide variety of higher plantspecies are well known and described in the technical, scientific, andpatent literature. See, for example, Weising et al., Ann. Rev. Genet.22:421-477 (1988). For example, the DNA construct may be introduceddirectly into the genomic DNA of the plant cell using techniques such aselectroporation, PEG poration, particle bombardment, silicon fiberdelivery, or microinjection of plant cell protoplasts or embryogeniccallus. See, e.g., Tomes et al., Direct DNA Transfer into Intact PlantCells Via Microprojectile Bombardment. pp.197-213 in Plant Cell, Tissueand Organ Culture, Fundamental Methods, Eds. O. L. Gamborg and G. C.Phillips, Springer-Verlag Berlin Heidelberg New York, 1995.Alternatively, the DNA constructs may be combined with suitable T-DNAflanking regions and introduced into a conventional Agrobacteriumtumefaciens host vector. The virulence functions of the Agrobactenumtumefaciens host will direct the insertion of the construct and adjacentmarker into the plant cell DNA when the cell is infected by thebacteria. See, U.S. Pat. No. 5,591,616.

[0120] The introduction of DNA constructs using polyethylene glycolprecipitation is described in Paszkowski et al., Embo J. 3:2717-2722(1984). Electroporation techniques are described in Fromm et al., Proc.Natl. Acad. Sci. U.S.A. 82:5824 (1985). Ballistic transformationtechniques are described in Klein et al., Nature 327:70-73 (1987).

[0121]Agrobacterium tumefaciens-meditated transformation techniques arewell described in the scientific literature. See, for example Horsch etal., Science 233:496-498 (1984), and Fraley et al., Proc. Natl. Acad.Sci. 80:4803 (1983). For instance, Agrobacterium transformation of maizeis described in U.S. Pat. No. 5,981,840. Agrobacterium transformation ofsoybean is described in U.S. Pat. No. 5,563,055.

[0122] Other methods of transformation include (1) Agrobacteriumrhizogenes-mediated transformation (see, e.g., Lichtenstein and FullerIn: Genetic Engineering, Vol. 6, P. W. J. Rigby, Ed., London, AcademicPress, 1987; and Lichtenstein, C. P. and Draper, J. In: DNA Cloning,Vol. II, D. M. Glover, Ed., Oxford, IRI Press, 1985), ApplicationPCT/US87/02512 (WO 88/02405 published Apr. 7, 1988) describes the use ofA. rhizogenes strain A4 and its Ri plasmid along with A. tumefaciensvectors pARC8 or pARC16, (2) liposome-mediated DNA uptake (see, e.g.,Freeman et al., Plant Cell Physiol. 25:1353 (1984)), and (3) thevortexing method (see, e.g., Kindle, Proc. Natl. Acad. Sci. USA 87:1228(1990)).

[0123] DNA can also be introduced into plants by direct DNA transferinto pollen as described by Zhou et al., Methods in Enzymology, 101:433(1983); D. Hess, Intern Rev. Cytol., 107:367 (1987); Luo et al., PlantMol. Biol. Reporter, 6:165 (1988). Expression of polypeptide codingpolynucleotides can be obtained by injection of the DNA intoreproductive organs of a plant as described by Pena et al., Nature,325:274 (1987). DNA can also be injected directly into the cells ofimmature embryos and the rehydration of desiccated embryos as describedby Neuhaus et al., Theor. Appl. Genet. 75:30 (1987); and Benbrook etal., in Proceedings Bio Expo 1986, Butterworth, Stoneham, Mass., pp.27-54 (1986).

[0124] Animal and lower eukaryotic (e.g., yeast) host cells arecompetent or rendered competent for transformation by various means.There are several well-known methods of introducing DNA into animalcells. These include: calcium phosphate precipitation, fusion of therecipient cells with bacterial protoplasts containing the DNA, treatmentof the recipient cells with liposomes containing the DNA, DEAE dextran,electroporation, biolistics, and micro-injection of the DNA directlyinto the cells. The transfected cells are cultured by means well knownin the art. Kuchler, R. J., Biochemical Methods in Cell Culture andVirology, Dowden, Hutchinson and Ross, Inc. (1977).

Transgenic Plant Regeneration

[0125] Transformed plant cells which are derived by any of the abovetransformation techniques can be cultured to regenerate a whole plantwhich possesses the transformed genotype. Such regeneration techniquesoften rely on manipulation of certain phytohormones in a tissue culturegrowth medium, typically relying on a biocide and/or herbicide markerthat has been introduced together with a polynucleotide of the presentinvention. For transformation and regeneration of maize see, Gordon-Kammet al., The Plant Cell 2:603-618 (1990).

[0126] Plants cells transformed with a plant expression vector can beregenerated, e.g., from single cells, callus tissue or leaf discsaccording to standard plant tissue culture techniques. It is well knownin the art that various cells, tissues, and organs from almost any plantcan be successfully cultured to regenerate an entire plant. Plantregeneration from cultured protoplasts is described in Evans et al.,Protoplasts Isolation and Culture, Handbook of Plant Cell Culture,Macmillan Publishing Company, New York, pp. 124-176 (1983); and Binding,Regeneration of Plants, Plant Protoplasts, CRC Press, Boca Raton, pp.21-73 (1985).

[0127] The regeneration of plants containing the foreign gene introducedby Agrobacterium can be achieved as described by Horsch et al., Science,227:1229-1231 (1985) and Fraley et al., Proc. Natl. Acad. Sci. U.S.A.80:4803 (1983). This procedure typically produces shoots within two tofour weeks and these transformant shoots are then transferred to anappropriate root-inducing medium containing the selective agent and anantibiotic to prevent bacterial growth. Transgenic plants of the presentinvention may be fertile or sterile.

[0128] Regeneration can also be obtained from plant callus, explants,organs, or parts thereof. Such regeneration techniques are describedgenerally in Klee et al., Ann. Rev. of Plant Phys. 38:467-486 (1987).The regeneration of plants from either single plant protoplasts orvarious explants is well known in the art. See, for example, Methods forPlant Molecular Biology, A. Weissbach and H. Weissbach, eds., AcademicPress, Inc., San Diego, Calif. (1988). For maize cell culture andregeneration see generally, The Maize Handbook, Freeling and Walbot,Eds., Springer, N.Y. (1994); Corn and Corn Improvement, 3^(rd) edition,Sprague and Dudley Eds., American Society of Agronomy, Madison, Wis.(1988).

[0129] One of skill will recognize that after the expression cassette isstably incorporated in transgenic plants and confirmed to be operable,it can be introduced into other plants by sexual crossing. Any of anumber of standard breeding techniques can be used, depending upon thespecies to be crossed.

[0130] In vegetatively propagated crops, mature transgenic plants can bepropagated by the taking of cuttings, via production of apomictic seed,or by tissue culture techniques to produce multiple identical plants.Selection of desirable transgenics is made and new varieties areobtained and propagated vegetatively for commercial use. In seedpropagated crops, mature transgenic plants can be self crossed toproduce a homozygous inbred plant. The inbred plant produces seedcontaining the newly introduced heterologous nucleic acid. These seedscan be grown to produce plants that would produce the selectedphenotype.

[0131] Parts obtained from the regenerated plant, such as flowers,seeds, leaves, branches, fruit, and the like are included in theinvention, provided that these parts comprise cells comprising theisolated nucleic acid of the present invention. Progeny and variants,and mutants of the regenerated plants are also included within the scopeof the invention, provided that these parts comprise the introducednucleic acid sequences.

[0132] Transgenic plants expressing a selectable marker can be screenedfor transmission of the nucleic acid of the present invention by, forexample, standard immunoblot and DNA detection techniques. Transgeniclines are also typically evaluated on levels of expression of theheterologous nucleic acid. Expression at the RNA level can be determinedinitially to identify and quantitate expression-positive plants.Standard techniques for RNA analysis can be employed and include PCRamplification assays using oligonucleotide primers designed to amplifyonly the heterologous RNA templates and solution hybridization assaysusing heterologous nucleic acid-specific probes. The RNA-positive plantscan then be analyzed for protein expression by Western immunoblotanalysis using the specifically reactive antibodies of the presentinvention. In addition, in situ hybridization and immunocytochemistryaccording to standard protocols can be done using heterologous nucleicacid specific polynucleotide probes and antibodies, respectively, tolocalize sites of expression within transgenic tissue. Generally, anumber of transgenic lines are usually screened for the incorporatednucleic acid to identify and select plants with the most appropriateexpression profiles.

[0133] Transgenic plants of the present invention can be homozygous forthe added heterologous nucleic acid; i.e., a transgenic plant thatcontains two added nucleic acid sequences, one gene at the same locus oneach chromosome of a chromosome pair. A homozygous transgenic plant canbe obtained by sexually mating (selfing) a heterozygous transgenic plantthat contains a single added heterologous nucleic acid, germinating someof the seed produced and analyzing the resulting plants produced foraltered expression of a polynucleotide of the present invention relativeto a control plant (i.e., native, non-transgenic). Back-crossing to aparental plant and out-crossing with a non-transgenic plant are alsocontemplated. Alternatively, propagation of heterozygous transgenicplants could be accomplished through apomixis.

[0134] The present invention provides a method of genotyping a plantcomprising a polynucleotide of the present invention. Genotypingprovides a means of distinguishing homologs of a chromosome pair and canbe used to differentiate segregants in a plant population. Molecularmarker methods can be used for phylogenetic studies, characterizinggenetic relationships among crop varieties, identifying crosses orsomatic hybrids, localizing chromosomal segments affecting monogenictraits, map based cloning, and the study of quantitative inheritance.See, e.g., Plant Molecular Biology: A Laboratory Manual, Chapter 7,Clark, Ed., Springer-Verlag, Berlin (1997). For molecular markermethods, see generally, The DNA Revolution by Andrew H. Paterson 1996(Chapter 2) in: Genome Mapping in Plants (ed. Andrew H. Paterson) byAcademic Press/R. G. Landis Company, Austin, Tex., pp.7-21.

[0135] The particular method of genotyping in the present invention mayemploy any number of molecular marker analytic techniques such as, butnot limited to, restriction fragment length polymorphisms (RFLPs). RFLPsare the product of allelic differences between DNA restriction fragmentscaused by nucleotide sequence variability. Thus, the present inventionfurther provides a means to follow segregation of a gene or nucleic acidof the present invention as well as chromosomal sequences geneticallylinked to these genes or nucleic acids using such techniques as RFLPanalysis.

[0136] Plants which can be used in the method of the invention includemonocotyledonous and dicotyledonous plants. Preferred plants includemaize, wheat, rice, barley, oats, sorghum, millet, rye, soybean,sunflower, safflower, alfalfa, canola, cotton, or turf grass.

[0137] Seeds derived from plants regenerated from transformed plantcells, plant parts or plant tissues, or progeny derived from theregenerated transformed plants, may be used directly as feed or food, orfurther processing may occur.

[0138] All publications cited in this application are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

[0139] The present invention will be further described by reference tothe following detailed examples. It is understood, however, that thereare many extensions, variations, and modifications on the basic theme ofthe present invention beyond that shown in the examples and description,which are within the spirit and scope of the present invention.

[0140] Other objects, features, advantages and aspects of the presentinvention will become apparent to those of skill from the followingdescription. It should be understood, however, that the followingdescription and the specific examples, while indicating certainembodiments of the invention, are given by way of illustration only.Various changes and modifications within the spirit and scope of thedisclosed invention will become readily apparent to those skilled in theart from reading the following description and from reading the otherparts of the present disclosure.

EXAMPLES Example 1 cDNA Library Construction

[0141] A. Total RNA Isolation

[0142] Total RNA was isolated from maize tissues with TRIzol Reagent(Life Technology Inc. Gaithersburg, Md.) using a modification of theguanidine isothiocyanate/acid-phenol procedure described by Chomczynskiand Sacchi (Anal. Biochem. 162, 156 (1987)). In brief, plant tissuesamples were pulverized in liquid nitrogen before the addition of theTRIzol Reagent, and then were further homogenized with a mortar andpestle. Addition of chloroform followed by centrifugation was conductedfor separation of an aqueous phase and an organic phase. The total RNAwas recovered by precipitation with isopropyl alcohol from the aqueousphase.

[0143] B. Poly(A)+ RNA Isolation

[0144] The selection of poly(A)+ RNA from total RNA was performed usingPolyATract system (Promega Corporation. Madison, Wis.). In brief,biotinylated oligo(dT) primers were used to hybridize to the 3′ poly(A)tails on mRNA. The hybrids were captured using streptavidin coupled toparamagnetic particles and a magnetic separation stand. The mRNA waswashed at high stringent condition and eluted by RNase-free deionizedwater.

[0145] C. cDNA Library Construction

[0146] cDNA synthesis was performed and unidirectional cDNA librarieswere constructed using the SuperScript Plasmid System (Life TechnologyInc. Gaithersburg, Md.). The first stand of cDNA was synthesized bypriming an oligo(dT) primer containing a Not I site. The reaction wascatalyzed by SuperScript Reverse Transcriptase II at 45° C. The secondstrand of cDNA was labeled with alpha-³²P-dCTP and a portion of thereaction was analyzed by agarose gel electrophoresis to determine cDNAsizes. cDNA molecules smaller than 500 base pairs and unligated adapterswere removed by Sephacryl-S400 chromatography. The selected cDNAmolecules were ligated into pSPORT1 vector in between Not I and Sal Isites.

Example 2 Sequencing and cDNA Subtraction Procedures Used for MaizeEST's

[0147] A. Sequencing Template Preparation

[0148] Individual colonies were picked and DNA was prepared either byPCR with M13 forward primers and M13 reverse primers, or by plasmidisolation. All the cDNA clones were sequenced using M13 reverse primers.

[0149] B. Q-bot Subtraction Procedure

[0150] cDNA libraries subjected to the subtraction procedure were platedout on 22×22 cm² agar plate at density of about 3,000 colonies perplate. The plates were incubated in a 37° C. incubator for 12-24 hours.Colonies were picked into 384-well plates by a robot colony picker,Q-bot (GENETIX Limited). These plates were incubated overnight at 37° C.

[0151] Once sufficient colonies were picked, they were pinned onto 22×22cm² nylon membranes using Q-bot. Each membrane contained 9,216 coloniesor 36,864 colonies. These membranes were placed onto individual agarplates with appropriate antibiotic. The plates were incubated at 37° C.for overnight.

[0152] After colonies were recovered on the second day, these filterswere placed on filter paper prewetted with denaturing solution for fourminutes, then were incubated on top of a boiling water bath foradditional four minutes. The filters were then placed on filter paperprewetted with neutralizing solution for four minutes. After excesssolution was removed by placing the filters on dry filter papers for oneminute, the colony side of the filters were place into Proteinase Ksolution, incubated at 37° C. for 40-50 minutes. The filters were placedon dry filter papers to dry overnight. DNA was then cross-linked tonylon membrane by UV light treatment.

[0153] Colony hybridization was conducted as described by Sambrook, J.,Fritsch, E. F. and Maniatis, T., (in Molecular Cloning: A LaboratoryManual, 2^(nd) Edition). The following probes were used in colonyhybridization:

[0154] 1. First strand cDNA from the same tissue from which the librarywas made to remove the most redundant clones.

[0155] 2. 48-192 most redundant cDNA clones from the same library basedon previous sequencing data.

[0156] 3. 192 most redundant cDNA clones in the entire corn sequencedatabase.

[0157] 4. A Sal-A20 oligonucleotide: TCG ACC CAC GCG TCC GM AAA AAAM AAAAAA AAA, removes clones containing a poly A tail but no cDNA. See SEQ IDNO: 28.

[0158] 5. cDNA clones derived from rRNA.

[0159] The image of the autoradiography was scanned into computer andthe signal intensity and cold colony addresses of each colony wasanalyzed. Re-arraying of cold-colonies from 384 well plates to 96 wellplates was conducted using Q-bot.

Example 3 Identification and Isolation of IPPK Genes Using PCR

[0160] The presence of the IPPK polynucleotide is analyzed by PCR usingthe commercially available Roche Expand High Fidelity PCR System.Template DNA was isolated using the CTAB method of Example 5C. Theprimers of SEQ ID NOS: 26 and 27 were used to amplify the gene ofinterest from various maize lines. The buffer and polymeraseconcentrations were used as defined for the kit with the DNAconcentrations and cycling conditions as follows:

[0161] DNA concentrations:

[0162] 500 ng template DNA and 0.3 μM primers in a 50 μl PCR reactionmixture containing 200 μM dNTPs in buffer and polymerase provided by theRoche kit.

[0163] Thermocycling conditions are as follows (#cycles):  1 cycle:denature 2 min. at 94° C. 10 cycles: denature 15 sec. at 94° C. anneal30 sec. at 55° C. elongate 60 sec. at 68° C. 15 cycles: denature 15 sec.at 94° C. anneal 30 sec at 55° C. elongate 60 sec. + 5 sec. each cycleat 68° C.  1 cycle: elongate 7 min. at 72° C.

[0164] The products of the PCR reaction were analyzed on agarose gelsusing standard molecular biology techniques.

Example 4 Vector Construction

[0165] All vectors were constructed using standard molecular biologytechniques used by those of skill in the art (Sambrook et al., supra).

[0166] A. Vectors for Plant Transformation

[0167] Vectors were constructed for plant transformation using eitherparticle bombardment or Agrobacterium transformation protocols. Plasmidswere constructed by inserting IPPK expression cassettes, including thefollowing: oleosin promoter::IPPK::nos terminator, oleosinpromoter::ubiquiton intron::IPPK::nos terminator, or globulin1promoter::IPPK::globulin1 terminator, into a descendent plasmid of pSB11which contains the BAR expression cassette. Both the IPPK and the BARexpression cassettes were located between the right and left borders ofthe T-DNA.

[0168] For example, the Zea mays IPPK coding region, including the 5′UTR and 3′ UTR was isolated from a full length EST clone as a 1.18 kbEcoRI/SapI fragment. The fragment was blunt ended using Klenow and thefragment inserted in frame into a EcoRV site of a plasmid between theoleosin promoter and the Nos terminator. Orientation was confirmed usinga restriction enzyme digest. The oleosin promoter::IPPK::nos terminatortranscription unit is flanked by BstEII sites which were used to excisethe fragment and insert it into a binary vector containing the BARselectable marker. The IPPK cassette is linked to the selectable markerbetween the right and left borders of the T-DNA. This vector was usedfor insert preparation for particle gun transformation as well as forgenerating Agrobacterium transformation vectors as described below. Inthis case, insert DNA for particle gun transformation was generated byisolating the 6.16 kb Pmel fragment from the vector.

[0169] The plasmid pSB11 was obtained from Japan Tobacco Inc. (Tokyo,Japan). The construction of pSB11 from pSB21 and the construction ofpSB21 from starting vectors is described by Komari et al. (1996, PlantJ. 10:165-174). The T-DNA of the plasmid was integrated in to thesuperbinary plasmid pSB1 (Saito et al. EP 672 752 A1) by homologousrecombination between the two plasmids. The plasmid pSB1 was alsoobtained from Japan Tobacco Inc. These plasmids were either used forparticle bombardment transformation, or for Agrobacterium-mediatedtransformation after making a cointegrate in an appropriateAgrobacterium strain as described more fully below.

[0170] Competent cells of the Agrobacterium strain LBA4404 harboringpSB1 were created using the protocol as described by Lin (1995) inMethods in Molecular Biology, ed. Nickoloff, J. A. (Humana Press,Totowa, N.J.). The plasmid containing the expression cassettes waselectroporated into competent cells of the Agrobacterium strain LBA4404harboring pSB1 to create the cointegrate plasmid in Agrobacterium. Cellsand DNA were prepared for electroporation by mixing 1 ul of plasmid DNA(˜100 ng) with 20 ul of competent Agrobacterium cells in a 0.2 cmelectrode gap cuvette (Bio-Rad Cat# 165-2086, Hercules, Calif.).Electroporation was performed in a Bio-Rad Micropulser (Cat# 165-2100,Hercules, Calif.) using the EC2 setting, which delivers 2.5 kV to thecells. Successful recombination was verified by restriction analysis ofthe plasmid after transformation of the cointegrate plasmid back into E.coli DH5α cells.

[0171] B. Vectors for In Vitro Protein Expression in E. coli

[0172] Vectors are constructed for protein expression of IPPKs (SEQ IDNOS: 1, 3, 5, 7, 9, 11, 13, 15, 20, 22, and 24) in E. coli usingstandard protocols. Each IPPK sequence can be fused with GST to produceGST-tagged proteins which can facilitate purification.

[0173] If needed, cloning sites are introduced into the IPPK sequencesby PCR. For example, a primer is designed which introduces a Smal siteto the 5′ end of the sequence, and another primer is designed whichintroduces a NotI site to the 3′ end of the sequence. Using theserestriction sites, the IPPK sequence is cloned into the pGEX-4T-2 vector(PHARMACIA BIOTECH) to generate the IPPK GST-tagged expression vector.

[0174] These expression vectors are used to transform E. coli strainDH5a using standard techniques. The expression of GST-tagged IPPKproteins and assay for substrate-specificity is further described inExample 7.

Example 5 Plant Transformation

[0175] A. Particle Bombardment Transformation and Regeneration of MaizeCallus

[0176] Immature maize embryos from greenhouse or field grown High typeII donor plants are bombarded with a plasmid containing an IPPKpolynucleotide of the invention operably linked to an appropriatepromoter. If the polynucleotide does not include a selectable marker,another plasmid containing a selectable marker gene can beco-precipitated on the particles used for bombardment. For example, aplasmid containing the PAT gene (Wohileben et al. (1988) Gene 70:25-37)which confers resistance to the herbicide Bialaphos can be used.Transformation is performed as follows.

[0177] The ears are surface sterilized in 50% Chlorox bleach plus 0.5%Micro detergent for 20 minutes, and rinsed two times with sterile water.The immature embryos are excised and placed embryo axis side down(scutellum side up), 25 embryos per plate. These are cultured on 560Lagar medium 4 days prior to bombardment in the dark. Medium 560L is anN6-based medium containing Eriksson's vitamins, thiamine, sucrose,2,4-D, and silver nitrate. The day of bombardment, the embryos aretransferred to 560Y medium for 4 hours and are arranged within the2.5-cm target zone. Medium 560Y is a high osmoticum medium (560L withhigh sucrose concentration).

[0178] A plasmid vector comprising a polynucleotide of the inventionoperably linked to the selected promoter is constructed. This plasmidDNA, plus plasmid DNA containing a PAT selectable marker if needed, isprecipitated onto 1.1 μm (average diameter) tungsten pellets using aCaCl₂ precipitation procedure as follows: 100 μl prepared tungstenparticles (0.6 mg) in water, 20 μl (2 μg) DNA in TrisEDTA buffer (1 μgtotal), 100 μl 2.5 M CaC1₂, 40 μl 0.1 M spermidine.

[0179] Each reagent is added sequentially to the tungsten particlesuspension. The final mixture is sonicated briefly. After theprecipitation period, the tubes are centrifuged briefly, liquid removed,washed with 500 ml 100% ethanol, and centrifuged again for 30 seconds.Again the liquid is removed, and 60 μl 100% ethanol is added to thefinal tungsten particle pellet. For particle gun bombardment, thetungsten/DNA particles are briefly sonicated and 5 μl spotted onto thecenter of each macrocarrier and allowed to dry about 2 minutes beforebombardment.

[0180] The sample plates are bombarded at a distance of 8 cm from thestopping screen to the tissue, using a DuPont biolistics helium particlegun. All samples receive a single shot at 650 PSI, with a total of tenaliquots taken from each tube of prepared particles/DNA.

[0181] Four to 12 hours post bombardment, the embryos are moved to 560P(a low osmoticum callus initiation medium similar to 560L but with lowersilver nitrate), for 3-7 days, then transferred to 560R selectionmedium, an N6 based medium similar to 560P containing 3 mg/literBialaphos, and subcultured every 2 weeks. After approximately 10 weeksof selection, callus clones are sampled for PCR and activity of thepolynucleotide of interest. Positive lines are transferred to 288Jmedium, an MS-based medium with lower sucrose and hormone levels, toinitiate plant regeneration. Following somatic embryo maturation (2-4weeks), well-developed somatic embryos are transferred to medium forgermination and transferred to the lighted culture room. Approximately7-10 days later, developing plantlets are transferred to medium in tubesfor 7-10 days until plantlets are well established. Plants are thentransferred to inserts in flats (equivalent to 2.5″ pot) containingpotting soil and grown for 1 week in a growth chamber, subsequentlygrown an additional 1-2 weeks in the greenhouse, then transferred toClassic™ 600 pots (1.6 gallon) and grown to maturity. Plants aremonitored for expression of the polynucleotide of interest.

[0182] B. Agrobacterium-mediated Transformation and Regeneration ofMaize Callus

[0183] For Agrobacterium-mediated transformation of maize, an IPPKnucleotide sequence of the present invention was introduced using themethod of Zhao (U.S. Pat. No. 5,981,840, and PCT patent publicationWO98/32326; the contents of which are hereby incorporated by reference).

[0184] Briefly, immature embryos were isolated from maize and theembryos contacted with a suspension of Agrobacterium containing apolynucleotide of the present invention, where the bacteria are capableof transferring the nucleotide sequence of interest to at least one cellof at least one of the immature embryos (step 1: the infection step). Inthis step the immature embryos were immersed in an Agrobacteriumsuspension for the initiation of inoculation. The embryos wereco-cultured for a time with the Agrobacterium (step 2: theco-cultivation step). The immature embryos were cultured on solid mediumfollowing the infection step. Following this co-cultivation period anoptional “resting” step is contemplated. In this resting step, theembryos were incubated in the presence of at least one antibiotic knownto inhibit the growth of Agrobacterium without the addition of aselective agent for plant transformants (step 3: resting step). Theimmature embryos were cultured on solid medium with antibiotic, butwithout a selecting agent, for elimination of Agrobacterium and for aresting phase for the infected cells. Next, inoculated embryos werecultured on medium containing a selective agent and growing transformedcallus was recovered (step 4: the selection step). The immature embryoswere cultured on solid medium with a selective agent resulting in theselective growth of transformed cells. The callus was then regeneratedinto plants (step 5: the regeneration step), and calli grown onselective medium were cultured on solid medium to regenerate the plants.

[0185] C. Transformation of Dicots with Transgene

[0186] An expression cassette, with a polynucleotide of the presentinvention operably linked to appropriate regulatory elements forexpression can be introduced into embryogenic suspension cultures ofsoybean by particle bombardment using essentially the methods describedin Parrott, W. A., L. M. Hoffman, D. F. Hildebrand, E. G. Williams, andG. B. Collins, (1989) Recovery of primary transformants of soybean,Plant Cell Rep. 7:615-617. This method, with modifications, is describedbelow.

[0187] Seed is removed from pods when the cotyledons are between 3 and 5mm in length. The seeds are sterilized in a bleach solution (0.5%) for15 minutes after which time the seeds are rinsed with sterile distilledwater. The immature cotyledons are excised by first cutting away theportion of the seed that contains the embryo axis. The cotyledons arethen removed from the seed coat by gently pushing the distal end of theseed with the blunt end of the scalpel blade. The cotyledons are thenplaced (flat side up) SB1 initiation medium (MS salts, B5 vitamins, 20mg/L 2,4-D, 31.5 g/l sucrose, 8 g/L TC Agar, pH 5.8). The Petri platesare incubated in the light (16 hr day; 75-80 μE) at 26° C. After 4 weeksof incubation the cotyledons are transferred to fresh SB1 medium. Afteran additional two weeks, globular stage somatic embryos that exhibitproliferative areas are excised and transferred to FN Lite liquid medium(Samoylov, V. M., D. M. Tucker, and W. A. Parrott (1998) Soybean[Glycine max (L.) Merrill] embryogenic cultures: the role of sucrose andtotal nitrogen content on proliferation. In Vitro Cell Dev. Biol.-Plant34:8-13). About 10 to 12 small clusters of somatic embryos are placed in250 ml flasks containing 35 ml of SB172 medium. The soybean embryogenicsuspension cultures are maintained in 35 mL liquid media on a rotaryshaker, 150 rpm, at 26° C. with florescent lights (20 μE) on a 16:8 hourday/night schedule. Cultures are sub-cultured every two weeks byinoculating approximately 35 mg of tissue into 35 mL of liquid medium.

[0188] Soybean embryogenic suspension cultures are then transformedusing particle gun bombardment (Klein et al. (1987) Nature (London)327:70, U.S. Pat. No. 4,945,050). A BioRad Biolistic™ PDS1000/HEinstrument can be used for these transformations. A selectable markergene, which is used to facilitate soybean transformation, is a chimericgene composed of the 35S promoter from Cauliflower Mosaic Virus (Odellet al. (1985) Nature 313:810-812), the hygromycin phosphotransferasegene from plasmid pJR225 (from E. coli; Gritz et al. (1983) Gene25:179-188) and the 3′ region of the nopaline synthase gene from theT-DNA of the Ti plasmid of Agrobacterium tumefaciens.

[0189] To 50 μL of a 60 mg/mL 1 μm gold particle suspension is added (inorder): 5 μL DNA (1 μg/μL), 20 μl spermidine (0.1 M), and 50 μL CaCl₂(2.5 M). The particle preparation is agitated for three minutes, spun ina microfuge for 10 seconds and the supernatant removed. The DNA-coatedparticles are washed once in 400 μL 70% ethanol and resuspended in 40 μLof anhydrous ethanol. The DNA/particle suspension is sonicated threetimes for one second each. Five μL of the DNA-coated gold particles arethen loaded on each macro carrier disk.

[0190] Approximately 300-400 mg of a two-week-old suspension culture isplaced in an empty 60×15 mm petri dish and the residual liquid removedfrom the tissue with a pipette. Membrane rupture pressure is set at 1100psi and the chamber is evacuated to a vacuum of 28 inches mercury. Thetissue is placed approximately 8 cm away from the retaining screen, andis bombarded three times. Following bombardment, the tissue is dividedin half and placed back into 35 ml of FN Lite medium.

[0191] Five to seven days after bombardment, the liquid medium isexchanged with fresh medium. Eleven days post bombardment the medium isexchanged with fresh medium containing 50 mg/mL hygromycin. Thisselective medium is refreshed weekly. Seven to eight weeks postbombardment, green, transformed tissue will be observed growing fromuntransformed, necrotic embryogenic clusters. Isolated green tissue isremoved and inoculated into individual flasks to generate new, clonallypropagated, transformed embryogenic suspension cultures. Each new lineis treated as an independent transformation event. These suspensions arethen subcultured and maintained as clusters of immature embryos, ortissue is regenerated into whole plants by maturation and germination ofindividual embryos.

[0192] D. DNA Isolation from Callus and Leaf Tissues

[0193] In order to screen putative transformation events for thepresence of the transgene, genomic DNA is extracted from calluses orleaves using a modification of the CTAB (cetyltriethylammonium bromide,Sigma H5882) method described by Stacey and Isaac (1994). Approximately100-200 mg of frozen tissues is ground into powder in liquid nitrogenand homogenised in 1 ml of CTAB extraction buffer (2% CTAB, 0.02 M EDTA,0.1 M Tris-Cl pH 8, 1.4 M NaCl, 25 mM DTT) for 30 min at 65° C.Homogenised samples are allowed to cool at room temperature for 15 minbefore a single protein extraction with approximately 1 ml 24:1 v/vchloroform:octanol is done. Samples are centrifuged for 7 min at 13,000rpm and the upper layer of supernatant collected using wide-mouthedpipette tips. DNA is precipitated from the supernatant by incubation in95% ethanol on ice for 1 h. DNA threads are spooled onto a glass hook,washed in 75% ethanol containing 0.2 M sodium acetate for 10 min,air-dried for 5 min and resuspended in TE buffer. Five μl RNAse A isadded to the samples and incubated at 37° C. for 1 h.

[0194] For quantification of genomic DNA, gel electrophoresis isperformed using a 0.8% agarose gel in 1× TBE buffer. One microlitre ofthe samples are fractionated alongside 200, 400, 600 and 800 ng μl¹ λuncut DNA markers.

Example 6 Identification of High Phosphorus/Low Phytate Transgenic CornLines

[0195] The resulting transformants are screened for inorganic phosphorusand/or phytate levels using the calorimetric assays as described below.The extraction procedure used is compatible with both assays. Thecalorimetric assays can be performed sequentially or simultaneously.Putative events are usually initially screened for increased levels ofinorganic phosphorous compared to wild type control and then furthercharacterized by the phytate assay.

[0196] A. Sample Preparation

[0197] Individual kernels are crushed to a fine powder using a ball millgrinding device. Grinding of certain samples, for example high oil cornlines, can be facilitated by chilling the sample in the grindingapparatus at −80° C. for 2 hours prior to grinding. Transfer 25-35 mg ofeach ground sample to new 1.5 ml microfuge tube. Extract each samplewith 1 ml of 0.4N hydrochloric acid (HCl) for 3.5 hours at roomtemperature with shaking to keep the meal suspended. Transfer 1 ml ofthis suspension to a 1.1 ml Megatiter tube (Cat# 2610, Continental Labs)and place into the 96-well Megatiter plate (Cat# 2405, ContinentalLabs). Clarify the extract by low-speed centrifugation, for example 4000rpm for 15 minutes in a Jouan centrifuge. The clarified supernatant isused for the assays described in sections 6B and 6C below.

[0198] B. Quantitative Inorganic Phosphate Assay

[0199] This assay is performed in duplicate for each sample. For eachsample mix a 200 ul aliquot of clarified extract with 100 μl 30%trichloroacetic acid (TCA). Clarify by low speed centrifugation.Transfer 50 μl clarified supernatant to a 96-well microtiter plate. Add100 μl of the color reagent (7 parts 0.42% ammonium molybdate in 1NH2SO4: 1 part 10% ascorbic acid) and incubate at 37° C. for 30 minutes.A phosphate standard curve is generated using NaH₂PO₄ in the range of0-160 nmol diluted from a 10 mM stock solution in 2 parts 0.4N HCl: 1part 30% TCA. Measure the absorbance at 800 nm.

[0200] C. Quantitative Phytate Assay

[0201] This assay is modified from Haug and Lantzsch (1983) J. Sci. FoodAgric. 34:1423-1426. This assay is performed in duplicate for eachsample. Phytate standard (Cat# P-7660, Sigma Chemical Co., St. Louis,Mo.) stock solution is made by dissolving 150 mg phytate in 100 mldistilled water (DDW). Standards in the range of 0-35 μg/ml are made bydilution with 0.2N HCl. Samples are prepared in 96-well microtiterplates by mixing 35 μl of clarified supernatant (from 6A) with 35 μl ofDDW, add 140 μl ferric solution (0.2 g ammonium iron (III) sulphatedodecahydrate (Merck Art 3776)/liter in 0.2N HCl). Plates are sealed andincubated for 30 minutes at 99° C., then cooled to 4° C. Plates are keptin an ice-water bath for 15 minutes then transferred to room temperaturefor 20 minutes. Centrifuge the plates at low speed to pelletprecipitate, for example spin 30 minutes at 4000 rpm in a Jouancentrifuge. After centrifugation transfer 80 μl clarified supernatant toa new 96-well plate and mix with 120 μl 2,2′-bipyridine solution (10 g2,2′-bipyridine (Merck Art. 3098), 10 ml thioglycolic acid (Merck Art.700) in DDW to 1 liter).

[0202] Each plant identified as a potential high phosphorus transgenicis tested again to confirm the original elevated phosphorus reading.Confirmed high phosphorous lines are selected on the basis of uniformityfor the trait. Transformants which are positive with the colorimetricassays will then be subjected to more rigorous analyses to includeSouthern, Northern and Western blotting and/or quantitation andidentification of phytic acid and inositol phosphate intermediates byHPLC.

Example 7 Determining the Substrate Specificity of the ITPK clones

[0203] A. Expression of IPPK and Purification

[0204] A single colony of E. coli strain DH5α containing a GST-taggedITPK expression vector described in Example 4 is cultured overnight at37° C. in LB medium containing ampicillin (Amp). The overnight cultureis diluted 1:10 with fresh LB+Amp and incubated at 37° C. with vigorousagitation until the A600 reading of the culture is in the range of 1-2O.D. units. GST fusion protein expression is induced by the addition ofIPTG to the culture to a final concentration of 1 μM. The cultures areincubated at 37° C. with agitation for an additional 3 hrs.

[0205] Cells are harvested by centrifugation at 7,700×g for 10 minutesat 4° C. The cells are lysed on ice by sonication and the lystate isclarified by centrifugation at 12,000×g for 10 minutes at 4° C. TheGST-IPPK proteins are affinity purified by batch absorption toGlutathione Sepharose 4B bead resin (Bulk GST Purification kit,Pharmacia Biotech) at a ratio of 1 ml bed volume of the 50% GlutathioneSepharose 4B slurry per 100 ml clarified lysate. Following theconditions detailed in the manufacturer's instructions, the beads arewashed and GST-tagged IPPK protein eluted with 10 mM reduced glutathionein 50 mM Tris-HCl (pH 8.0). After elution, glycerol is added to a finalconcentration of 50% and purified GST-IPPK proteins are stored in 50%glycerol at −20° C. The protein concentration is adjusted toapproximately 50 μl.

[0206] B. Assay for IPPK Activity and Substrate Specificity

[0207] Purified GST-IPPK fusion proteins are used in an inositolpolyphosphate kinase activity assay. The activity assay is performed ina volume of 25 μl. The assay mixture contains 20 mM HEPES, pH 7.2, 6 mMMgCl₂, 10 mM LiCl, 1 mM DTT, 40 μM inositol phosphate substrate, 40 μMATP, 0.5 μl γ-³²P-ATP (3000 Ci/mmol) and 5 μl enzyme per reaction. Thereaction mixture is incubated at 30° C., or room temperature, for 30minutes. The reaction is stopped by the addition of 2.8 μl stoppingsolution (3M HCl, 2M KH₂PO₄) to the 25 μl reaction. One microlitersamples of each reaction, along with inositol phosphate standards, areseparated on a polyethyleneimine (PEI)-cellulose thin layerchromatography plate (Merck) with 0.5M HCl according to Spencer et al.(In Methods in Inositide Research, (1990) pp. 39-43, Ed. R. F. Irvine,Raven Press, NY). After separation, the TLC plate was air-dried at 70°C., wrapped in plastic wrap and exposed to X-ray film to detect the³²P-labelled reaction products. The reaction products were quantified bycutting the spot out of the TLC plate and measuring the radioactivity ina liquid scintillation counter. The identity of the reaction product wasconfirmed by comparing the distance migrated to the migration of theinositol phosphate standard controls run on each TLC plate. Severalinositol phosphate substrates are tested to determine the substratespecificity of the IPPK enzymes. The other substrates tested under thesame conditions above are: Ins(1)P, Ins(2)P, Ins(4)P, Ins(1,4)P₂,Ins(4,5)P₂, Ins(1,3,4)P₃, Ins(3,4,5)P₃, Ins(1,4,5)P₃, Ins(3,4,5,6)P₄,Ins(1,3,4,6)P4, Ins(1,3,5,6)P₄, and Ins(1,3,4,5,6)P₅.

Example 8 ITPK Corn Knockout Mutants

[0208] Mu-tagged corn populations (TUSC) are screened for knockouts ofthe IPPK gene, using the primers specific to the IPPK sequence ofinterest paired with a Mu-primer in PCR reactions. Lines identified ashaving a Mu-insertion in the IPPK gene are screened by further assays.Kernels from these lines are screened for phytate and inorganicphosphate levels versus phytate mutants Lpa1 and Lpa2, as well as wildtype controls, using the assays described in Example 6.

Example 9 Myo-inositol Assay

[0209] Putative events can also be screened to determine the effect thetransgene may have on myo-inositol levels in the kernel using a gaschromatography/mass spectrometry method.

[0210] Briefly, 20 representative whole, mature, dry kernels are groundto a fine meal in a ball mill apparatus. Each sample is analyzed intriplicate. For extraction, three aliquots of 0.5 g meal for each sampleis extracted with 5 ml of 50% v/v ethyl alcohol (1:1 100% ethylalcohol:DDW) at room temperature for one hour with vigorous shaking. Theextract supernatant is decanted and filtered through a 0.45 μm syringefilter. The meal residue is re-extracted with 5 ml of fresh 50% ethanolfollowing the same procedure, combining the two filtrates. Each sampleis vortexed, and a 1 ml aliquot taken and evaporated to dryness in aSpeedVac at medium heat.

[0211] A myo-inositol standard stock of 10 mg/ml is made in doubledistilled water (DDW) which is used to make a 1 mg/ml standard solutionworking stock. Aliquots of 50μl, 100 μl, 200 μl and 300μl aretransferred to new tubes and evaporated to dryness in a SpeedVac asdescribed above. This calibration set covers a concentration range of 5μg/ml to 30 μg/ml of each component.

[0212] Thoroughly dried standards and samples are resuspended in 50 μlpyridine. To this, 50 μl of 100:1trimethylsilylimadazole-trimethylchlorosilane (TMSI-TMCS) is added toeach sample. Samples are compromised if a precipitate forms. Tubes aresealed, vortexed and incubated 15 min. at 60° C. After incubation, 1mlof 2,2,4-timethylpentane and 0.5 ml DDW are added. Vortex samples andcentrifuge at low speed (2000 rpm) for 5 minutes. The top, organic layeris transferred to a 2ml autosampler vial which can be stored at 4° C.until it can be analyzed.

[0213] Samples are analyzed on a Hewlett-Packard 5890/7673/5972 GasChromatography/Mass Spectrometry (GC/MS) apparatus using aHewlett-Packard 30 m×0.25 mm i.d.×0.25 μm film thickness 5 MS columnunder the following conditions: Inlet temperature: 250° C. InjectionVolume: 1 ml Split Ratio: Splitless Oven Temperature: 70° C. initial,hold for 2 min. Ramp at 25/min. to 170° C., hold for 0 min. Ramp at5/min. to 215° C., hold for 0 min. Hold for 5 min., for a 23.4 min.total run time Detector Temperature: 250° C. Carrier Gas: Helium, 36.6cm/sec at 70° (1 ml/min.)

[0214] Full scan (m/z 50-650), 5 min. data collection delay. Results arereported as μg/ml for the final sample analyzed by the GC/MS, thisconcentration is multiplied by a factor of 20 before using to calculateμg/g dry weight tissue. The moisture content of the mature kernels isdetermined from a separate aliquot of each experimental sample so thatthe results can be adjusted to a dry weight basis.

[0215] Myo-inositol levels are quantified as follows:$\frac{\mu \quad g\quad {myo}\text{-}\text{inositol}}{g\quad {dry}\quad {{wt}.\quad {tissue}}} = {\frac{\mu \quad g\quad ({X20})}{{ml}\quad {sample}} \times \frac{1\quad {ml}\quad {sample}}{1\quad {ml}\quad {extract}} \times \frac{10\quad {ml}\quad {extract}}{0.5\quad g\quad {tissue}}}$

Example 10 HPLC of Phytate and Inositol Phosphate Intermediates

[0216] Phosphorous and inositol phosphate intermediates associated withphytic acid in wheat, corn, and soybean seeds can be identified andquantitated using gradient anion-exchange chromatography HPLC withconductivity detection. Phytate and the intermediate inositol phosphatescan be identified using this method. However, the method practicedcurrently has been optimized for phytate, it is not optimized forquantitation of intermediate inositol phosphates. For other HPLCseparations of inositol phosphates see also Anonymous, (1990) “Analysisof inositol phosphates” Dionex Corp. Application Note AN 65; Xu, P.,Price, J., and Aggett, P. (1992) Progress in Food and Nutrition Science16:245262; Rounds, M. A. and Nielsen, S. S. (1993) J.Chromatogr653:148-152; and Trugo, L. and von Baer, D. (1998) Associationfor animal production,publication 93:1128. Inositolphosphatescanalso beidentified by thin-layer chromatographic methods, see for exampleSpencer, C. E. L et al. (1990) Ch. 4 in Methods in Inositide Research,Ed. R. F. Irving, Raven Press, Ltd., NY pp. 3943; and Hatzack, F. andRasmussen, S. K. (1999) J. Chromatogr B 736:221-229.

[0217] For anion-exchange HPLC, a phytic acid standard range of 0.25,0.5, 1.0, 2.0 and 3.0 mg/ml is prepared in 0.4M hydrochloric acid (HCl)from a 20 mg/ml working stock in 0.4M HCl. Seed samples are prepared bygrinding seeds to a fine meal in a ball mill grinding apparatus.Replicate aliquots are weighed and extracted in 0.4M HCl in a ratio of0.1 g meal/1 ml 0.4M HCl. Usually 5 ml 0.4M HCl is used to extract 0.5 gcorn or wheat meal while 15 ml 0.4M HCl is used to extract 1.5 g soymeal. After the addition of the extraction buffer, the samples areextracted with moderate-vigorous shaking for 2 hrs. at room temperature,then transferred to 4° C. overnight without shaking. The supernatantsfrom corn and wheat are clarified by low-speed centrifugation. Due tothe high fat content, the low-speed supernatant from soy sample extractsis further clarified by ultracentrifugation at 55,000 rpm at 4° C. for 1hour. After ultracentrifugation, the clear, middle layer is removed witha needle or extended tip disposable transfer pipette. Clarified samplesare filtered through a 0.45 μm syringe filter and stored at 4° C. untilanalysis. Just before analysis, an aliquot of each sample is filteredwith a Millipore Durapore ULTRAFREE-MC 0.22 μm centrifugal filter unit,or equivalent.

[0218] Samples are subjected to anion-exchange HPLC separation by alinear gradient of 0.06-0.118M sodium hydroxide (NaOH) in 1% isopropylalcohol on a Dionex OmniPac PAX-100 column at a flow rate of 1 ml/min.The total run time is 30 min. with data collection from 0 to 20 minutes.Signal collection is set at 0.5 Hz, detector units in μS, current at 300mA, with the Detection Stablilizer regulated at 30° C. and temperaturecompensation at 1.7.

[0219] Twenty-five microliters extract is loaded onto the column.Soybean samples appear to cause column performance deterioration,therefore it is helpful to interject short column cleaning run betweensamples. The cleaning run comprises a series of injections for 1M HCl,1M NaOH, and 90% acetonitrile.

[0220] The above examples are provided to illustrate the invention butnot to limit its scope. Other variants of the invention will be readilyapparent to one of ordinary skill in the art and are encompassed by theappended claims. All publications, patents, patent applications, andcomputer programs cited herein are hereby incorporated by reference.

1 37 1 1169 DNA Zea mays CDS (84)...(806) 1 aaaatctctt tctccgctgcgctgcaaacc caccgcttcc accatcgcca ctcgtcaccc 60 cttgctccca tagtccccat accatg ccc gac ctc cac ccg ccg gag cac caa 113 Met Pro Asp Leu His Pro ProGlu His Gln 1 5 10 gtc gcc ggt cac cgc gcc tcc gcc agc aag ctg ggc ccgctc atc gac 161 Val Ala Gly His Arg Ala Ser Ala Ser Lys Leu Gly Pro LeuIle Asp 15 20 25 ggc tcc ggc ctc ttc tac aag ccg ctc cag gcc ggc gac cgtggg gag 209 Gly Ser Gly Leu Phe Tyr Lys Pro Leu Gln Ala Gly Asp Arg GlyGlu 30 35 40 cac gag gtc gcc ttc tat gag gcg ttc tcc gcc cac gcc gcc gtcccg 257 His Glu Val Ala Phe Tyr Glu Ala Phe Ser Ala His Ala Ala Val Pro45 50 55 gcc cgc atc cga gac acc ttc ttc ccc cgg ttc cac ggc acg cga ctc305 Ala Arg Ile Arg Asp Thr Phe Phe Pro Arg Phe His Gly Thr Arg Leu 6065 70 ctc ccc acc gag gcg cag ccc ggg gag ccg cat ccg cac ctc gtc ctc353 Leu Pro Thr Glu Ala Gln Pro Gly Glu Pro His Pro His Leu Val Leu 7580 85 90 gac gac ctc ctc gcg ggg ttt gag gcg ccc tgc gtc gca gac atc aag401 Asp Asp Leu Leu Ala Gly Phe Glu Ala Pro Cys Val Ala Asp Ile Lys 95100 105 atc ggc gcc atc acg tgg cca ccg agt tcg ccg gag ccc tac atc gcc449 Ile Gly Ala Ile Thr Trp Pro Pro Ser Ser Pro Glu Pro Tyr Ile Ala 110115 120 aag tac ctc gcc aag gac cgc ggg acc acg agc gtt ctg ctc gga ttc497 Lys Tyr Leu Ala Lys Asp Arg Gly Thr Thr Ser Val Leu Leu Gly Phe 125130 135 cgc gtc ttg cgt ccg agt cgt cgg ccc cga ggg cgc cgt gtg gcg gac545 Arg Val Leu Arg Pro Ser Arg Arg Pro Arg Gly Arg Arg Val Ala Asp 140145 150 gga gcg ccc gga ggt gaa ggc tat gga cac cgt cgg cgt ccg ccg cgt593 Gly Ala Pro Gly Gly Glu Gly Tyr Gly His Arg Arg Arg Pro Pro Arg 155160 165 170 gct ccg gcg cta cgt gtc atc cgc ttg ccg acg agg gga tgg actgcg 641 Ala Pro Ala Leu Arg Val Ile Arg Leu Pro Thr Arg Gly Trp Thr Ala175 180 185 cgc tcg cgg cgg cgg tgt acg gag gaa aag gtg gag tct tgt cacagc 689 Arg Ser Arg Arg Arg Cys Thr Glu Glu Lys Val Glu Ser Cys His Ser190 195 200 tgc gcg agc tca agg cat ggt tgg agg agc aga ctc tgt tcc acttct 737 Cys Ala Ser Ser Arg His Gly Trp Arg Ser Arg Leu Cys Ser Thr Ser205 210 215 act cgg cgt cga ttc ttc tgg gct atg atg ctg ctg cag tcg cagcag 785 Thr Arg Arg Arg Phe Phe Trp Ala Met Met Leu Leu Gln Ser Gln Gln220 225 230 gcg gag gtg ggg gtg ggg taa cagtgaagct ggtggacttt gcccatgtgg836 Ala Glu Val Gly Val Gly * 235 240 ccgagggtga tggggtgatt gaccacaacttcctgggcga gctctgctag ctgatcaagt 896 tcgtttctga cattgttcca gagactccttagacgcagcc tttgggtcct tcttaagaga 956 ggatcctgac atttttgatt tgataacaaaggaagcactt tcagctgcaa aaaaagaaag 1016 cagcagtgag gatgaagatg acagtagtgaggaaagttcg gatgatgagc caacaaaagt 1076 tgaagaaaag aaggctccaa aagtatcagaaaacattgga tctgaggatg aatcttctga 1136 agacgagagt gataaagaca gtgaagagcctca 1169 2 240 PRT Zea mays 2 Met Pro Asp Leu His Pro Pro Glu His GlnVal Ala Gly His Arg Ala 1 5 10 15 Ser Ala Ser Lys Leu Gly Pro Leu IleAsp Gly Ser Gly Leu Phe Tyr 20 25 30 Lys Pro Leu Gln Ala Gly Asp Arg GlyGlu His Glu Val Ala Phe Tyr 35 40 45 Glu Ala Phe Ser Ala His Ala Ala ValPro Ala Arg Ile Arg Asp Thr 50 55 60 Phe Phe Pro Arg Phe His Gly Thr ArgLeu Leu Pro Thr Glu Ala Gln 65 70 75 80 Pro Gly Glu Pro His Pro His LeuVal Leu Asp Asp Leu Leu Ala Gly 85 90 95 Phe Glu Ala Pro Cys Val Ala AspIle Lys Ile Gly Ala Ile Thr Trp 100 105 110 Pro Pro Ser Ser Pro Glu ProTyr Ile Ala Lys Tyr Leu Ala Lys Asp 115 120 125 Arg Gly Thr Thr Ser ValLeu Leu Gly Phe Arg Val Leu Arg Pro Ser 130 135 140 Arg Arg Pro Arg GlyArg Arg Val Ala Asp Gly Ala Pro Gly Gly Glu 145 150 155 160 Gly Tyr GlyHis Arg Arg Arg Pro Pro Arg Ala Pro Ala Leu Arg Val 165 170 175 Ile ArgLeu Pro Thr Arg Gly Trp Thr Ala Arg Ser Arg Arg Arg Cys 180 185 190 ThrGlu Glu Lys Val Glu Ser Cys His Ser Cys Ala Ser Ser Arg His 195 200 205Gly Trp Arg Ser Arg Leu Cys Ser Thr Ser Thr Arg Arg Arg Phe Phe 210 215220 Trp Ala Met Met Leu Leu Gln Ser Gln Gln Ala Glu Val Gly Val Gly 225230 235 240 3 923 DNA Zea mays CDS (53)...(736) 3 accgcttcca ccatcgccactcgtcacccc ttgctcccat agtccccata cc atg ccc 58 Met Pro 1 gac ctc cac ccgccg gag cac caa gtc gcc ggt cac cgc gcc tcc gcc 106 Asp Leu His Pro ProGlu His Gln Val Ala Gly His Arg Ala Ser Ala 5 10 15 agc aag ccg ggc ccgctc atc gac ggc tcc ggc ctc ttc tac aag ccg 154 Ser Lys Pro Gly Pro LeuIle Asp Gly Ser Gly Leu Phe Tyr Lys Pro 20 25 30 ctc cag gcc ggc gac cgtggg gag cac gag gtc gct ttc tat gag gcg 202 Leu Gln Ala Gly Asp Arg GlyGlu His Glu Val Ala Phe Tyr Glu Ala 35 40 45 50 ttc tcc gcc cac gcc gccgtc ccg gcc cgc atc cga gac acc ttc ttc 250 Phe Ser Ala His Ala Ala ValPro Ala Arg Ile Arg Asp Thr Phe Phe 55 60 65 ccc cgg ttc cac ggc acg cgactc ctc ccc acc gag gcg cag ccc ggg 298 Pro Arg Phe His Gly Thr Arg LeuLeu Pro Thr Glu Ala Gln Pro Gly 70 75 80 gag ccg cat ccg cac ctc gtc ctcgac gac ctc ctc gcg gga ttt gag 346 Glu Pro His Pro His Leu Val Leu AspAsp Leu Leu Ala Gly Phe Glu 85 90 95 gcg ccc tgc gtc gca gac atc aag atcggc gcc atc acg tgg cca ccg 394 Ala Pro Cys Val Ala Asp Ile Lys Ile GlyAla Ile Thr Trp Pro Pro 100 105 110 agt tcg ccg gag ccc tac atc gcc aagtgc ctc gcc atg gac cgc ggg 442 Ser Ser Pro Glu Pro Tyr Ile Ala Lys CysLeu Ala Met Asp Arg Gly 115 120 125 130 acc acg agc gtt ctg ctc gga ttccgc gtc tcc ggc gtc cga gtc gtc 490 Thr Thr Ser Val Leu Leu Gly Phe ArgVal Ser Gly Val Arg Val Val 135 140 145 gtc ccc gag ggc gcc gtg tgg cggacg gag cgc ccg gag gtg aag gct 538 Val Pro Glu Gly Ala Val Trp Arg ThrGlu Arg Pro Glu Val Lys Ala 150 155 160 atg gac acc gtc ggc gtc cgc cgcgtg ctc cgg cgc tac gtg tca tcc 586 Met Asp Thr Val Gly Val Arg Arg ValLeu Arg Arg Tyr Val Ser Ser 165 170 175 gct tgc cga cga ggg gat gga ctgcgc gct cgc ggc ggc ggt gta cgg 634 Ala Cys Arg Arg Gly Asp Gly Leu ArgAla Arg Gly Gly Gly Val Arg 180 185 190 agg aaa agg tgg agt ctt gtc actgct gcg cga gct caa ggc gtg gtt 682 Arg Lys Arg Trp Ser Leu Val Thr AlaAla Arg Ala Gln Gly Val Val 195 200 205 210 cga gga gca gcc tct gtt ccactt cta ctc ggc gtc gat tct tct ggg 730 Arg Gly Ala Ala Ser Val Pro LeuLeu Leu Gly Val Asp Ser Ser Gly 215 220 225 cta tga tgctgctgcagtcgcagcag gcggaggtgg gggtggggta acagtgaagc 786 Leu * tggtggactttgcccatgtg gccgagggtg atggggtgat tgaccacaac ttcctgggcg 846 ggctctgctagctgatcaag ttcgtttctg acattgttcc agagactcct cagacgcagc 906 ctttgggtccttcttaa 923 4 227 PRT Zea mays 4 Met Pro Asp Leu His Pro Pro Glu His GlnVal Ala Gly His Arg Ala 1 5 10 15 Ser Ala Ser Lys Pro Gly Pro Leu IleAsp Gly Ser Gly Leu Phe Tyr 20 25 30 Lys Pro Leu Gln Ala Gly Asp Arg GlyGlu His Glu Val Ala Phe Tyr 35 40 45 Glu Ala Phe Ser Ala His Ala Ala ValPro Ala Arg Ile Arg Asp Thr 50 55 60 Phe Phe Pro Arg Phe His Gly Thr ArgLeu Leu Pro Thr Glu Ala Gln 65 70 75 80 Pro Gly Glu Pro His Pro His LeuVal Leu Asp Asp Leu Leu Ala Gly 85 90 95 Phe Glu Ala Pro Cys Val Ala AspIle Lys Ile Gly Ala Ile Thr Trp 100 105 110 Pro Pro Ser Ser Pro Glu ProTyr Ile Ala Lys Cys Leu Ala Met Asp 115 120 125 Arg Gly Thr Thr Ser ValLeu Leu Gly Phe Arg Val Ser Gly Val Arg 130 135 140 Val Val Val Pro GluGly Ala Val Trp Arg Thr Glu Arg Pro Glu Val 145 150 155 160 Lys Ala MetAsp Thr Val Gly Val Arg Arg Val Leu Arg Arg Tyr Val 165 170 175 Ser SerAla Cys Arg Arg Gly Asp Gly Leu Arg Ala Arg Gly Gly Gly 180 185 190 ValArg Arg Lys Arg Trp Ser Leu Val Thr Ala Ala Arg Ala Gln Gly 195 200 205Val Val Arg Gly Ala Ala Ser Val Pro Leu Leu Leu Gly Val Asp Ser 210 215220 Ser Gly Leu 225 5 923 DNA Zea mays CDS (53)...(922) 5 accgcttccaccatcgccac tcgtcacccc ttgctcccat agtccccata cc atg ccc 58 Met Pro 1 gacctc cac ccg ccg gag cac caa gtc gcc ggt cac cgc gcc tcc gcc 106 Asp LeuHis Pro Pro Glu His Gln Val Ala Gly His Arg Ala Ser Ala 5 10 15 agc aagccg ggc ccg ctc atc gac ggc tcc ggc ctc ttc tac aag ccg 154 Ser Lys ProGly Pro Leu Ile Asp Gly Ser Gly Leu Phe Tyr Lys Pro 20 25 30 ctc cag gccggc gac cgt ggg gag cac gag gtc gct ttc tat gag gcg 202 Leu Gln Ala GlyAsp Arg Gly Glu His Glu Val Ala Phe Tyr Glu Ala 35 40 45 50 ttc tcc gcccac gcc gcc gtc ccg gcc cgc atc cga gac acc ttc ttc 250 Phe Ser Ala HisAla Ala Val Pro Ala Arg Ile Arg Asp Thr Phe Phe 55 60 65 ccc cgg ttc cacggc acg cga ctc ctc ccc acc gag gcg cag ccc ggg 298 Pro Arg Phe His GlyThr Arg Leu Leu Pro Thr Glu Ala Gln Pro Gly 70 75 80 gag ccg cat ccg cacctc gtc ctc gac gac ctc ctc gcg gga ttt gag 346 Glu Pro His Pro His LeuVal Leu Asp Asp Leu Leu Ala Gly Phe Glu 85 90 95 gcg ccc tgc gtc gca gacatc aag atc ggc gcc atc acg tgg cca ccg 394 Ala Pro Cys Val Ala Asp IleLys Ile Gly Ala Ile Thr Trp Pro Pro 100 105 110 agt tcg ccg gag ccc tacatc gcc aag tgc ctc gcc atg gac cgc ggg 442 Ser Ser Pro Glu Pro Tyr IleAla Lys Cys Leu Ala Met Asp Arg Gly 115 120 125 130 acc acg agc gtt ctgctc gga ttc cgc gtc tcc ggc gtc cga gtc gtc 490 Thr Thr Ser Val Leu LeuGly Phe Arg Val Ser Gly Val Arg Val Val 135 140 145 ggc ccc gag ggc gccgtg tgg cgg acg gag cgc ccg gag gtg aag gcc 538 Gly Pro Glu Gly Ala ValTrp Arg Thr Glu Arg Pro Glu Val Lys Ala 150 155 160 atg gac acc gcc ggcgtc cgc cgc gtg ctc cgg cgc tac gtg tca tcc 586 Met Asp Thr Ala Gly ValArg Arg Val Leu Arg Arg Tyr Val Ser Ser 165 170 175 gtt gcc gac gag gggatg gac tgt gcg ctc gcc gcg gcg gtg tac gga 634 Val Ala Asp Glu Gly MetAsp Cys Ala Leu Ala Ala Ala Val Tyr Gly 180 185 190 gga aaa ggt gga gtcttg tca cag ctg cgc gag ctc aag gcg tgg ttc 682 Gly Lys Gly Gly Val LeuSer Gln Leu Arg Glu Leu Lys Ala Trp Phe 195 200 205 210 gag gag cag actctg ttc cac ttc tac tcg gcg tcg att ctt ctg ggc 730 Glu Glu Gln Thr LeuPhe His Phe Tyr Ser Ala Ser Ile Leu Leu Gly 215 220 225 tat gat gct gctgca gtc gca gca ggc gga ggt ggg ggt ggg gtg acg 778 Tyr Asp Ala Ala AlaVal Ala Ala Gly Gly Gly Gly Gly Gly Val Thr 230 235 240 gtg aag ctg gtggac ttt gcc cat gtg gcc gag ggt gat ggg gtg att 826 Val Lys Leu Val AspPhe Ala His Val Ala Glu Gly Asp Gly Val Ile 245 250 255 gac cac aac ttcctg ggc ggg ctc tgc tcg ctg atc aag ttc gtt tct 874 Asp His Asn Phe LeuGly Gly Leu Cys Ser Leu Ile Lys Phe Val Ser 260 265 270 gac att gtt ccagag act cct cag acg cag cct ttg ggt cct tct taa 922 Asp Ile Val Pro GluThr Pro Gln Thr Gln Pro Leu Gly Pro Ser * 275 280 285 g 923 6 289 PRTZea mays 6 Met Pro Asp Leu His Pro Pro Glu His Gln Val Ala Gly His ArgAla 1 5 10 15 Ser Ala Ser Lys Pro Gly Pro Leu Ile Asp Gly Ser Gly LeuPhe Tyr 20 25 30 Lys Pro Leu Gln Ala Gly Asp Arg Gly Glu His Glu Val AlaPhe Tyr 35 40 45 Glu Ala Phe Ser Ala His Ala Ala Val Pro Ala Arg Ile ArgAsp Thr 50 55 60 Phe Phe Pro Arg Phe His Gly Thr Arg Leu Leu Pro Thr GluAla Gln 65 70 75 80 Pro Gly Glu Pro His Pro His Leu Val Leu Asp Asp LeuLeu Ala Gly 85 90 95 Phe Glu Ala Pro Cys Val Ala Asp Ile Lys Ile Gly AlaIle Thr Trp 100 105 110 Pro Pro Ser Ser Pro Glu Pro Tyr Ile Ala Lys CysLeu Ala Met Asp 115 120 125 Arg Gly Thr Thr Ser Val Leu Leu Gly Phe ArgVal Ser Gly Val Arg 130 135 140 Val Val Gly Pro Glu Gly Ala Val Trp ArgThr Glu Arg Pro Glu Val 145 150 155 160 Lys Ala Met Asp Thr Ala Gly ValArg Arg Val Leu Arg Arg Tyr Val 165 170 175 Ser Ser Val Ala Asp Glu GlyMet Asp Cys Ala Leu Ala Ala Ala Val 180 185 190 Tyr Gly Gly Lys Gly GlyVal Leu Ser Gln Leu Arg Glu Leu Lys Ala 195 200 205 Trp Phe Glu Glu GlnThr Leu Phe His Phe Tyr Ser Ala Ser Ile Leu 210 215 220 Leu Gly Tyr AspAla Ala Ala Val Ala Ala Gly Gly Gly Gly Gly Gly 225 230 235 240 Val ThrVal Lys Leu Val Asp Phe Ala His Val Ala Glu Gly Asp Gly 245 250 255 ValIle Asp His Asn Phe Leu Gly Gly Leu Cys Ser Leu Ile Lys Phe 260 265 270Val Ser Asp Ile Val Pro Glu Thr Pro Gln Thr Gln Pro Leu Gly Pro 275 280285 Ser 7 1344 DNA Zea mays CDS (52)...(921) 7 gcacgaggtc agtccgtcacccctcgcgcc catagtcccc ttccccatac c atg tcc 57 Met Ser 1 gac ctc cac ccgccg gag cac caa gtc gcc ggc cac cgc gcc tcc gcc 105 Asp Leu His Pro ProGlu His Gln Val Ala Gly His Arg Ala Ser Ala 5 10 15 agc aag ctg ggc ccgctc atc gac ggc tcc ggc ctc ttc tac aag ccg 153 Ser Lys Leu Gly Pro LeuIle Asp Gly Ser Gly Leu Phe Tyr Lys Pro 20 25 30 ctc cag gcc ggc gac cgtggg gag cac gag gtc gcc ttc tat gag gcg 201 Leu Gln Ala Gly Asp Arg GlyGlu His Glu Val Ala Phe Tyr Glu Ala 35 40 45 50 ttc tcc gcc cac gcc gccgtc ccg gcc cgc atc cga gac acc ttc ttc 249 Phe Ser Ala His Ala Ala ValPro Ala Arg Ile Arg Asp Thr Phe Phe 55 60 65 ccc cgg ttc cac ggc acg cgactc ctc ccc acc gag gcg cag ccc ggg 297 Pro Arg Phe His Gly Thr Arg LeuLeu Pro Thr Glu Ala Gln Pro Gly 70 75 80 gag ccg cat cct cac ctc gtc ctcgac gac ctc ctc gcg ggg ttt cag 345 Glu Pro His Pro His Leu Val Leu AspAsp Leu Leu Ala Gly Phe Gln 85 90 95 gcg ccc tgc gtc gca gac atc aag atcggc gcc atc acg tgg cca ccg 393 Ala Pro Cys Val Ala Asp Ile Lys Ile GlyAla Ile Thr Trp Pro Pro 100 105 110 agt tcg ccg gag ccc tac atc gcc aagtgc ctc gcc aag gac cgc ggg 441 Ser Ser Pro Glu Pro Tyr Ile Ala Lys CysLeu Ala Lys Asp Arg Gly 115 120 125 130 acc acg agc gtt ctg ctc gga ttccgc gtc tcc ggc gtc cga gtc gtc 489 Thr Thr Ser Val Leu Leu Gly Phe ArgVal Ser Gly Val Arg Val Val 135 140 145 ggc ccc gag ggc gcc gtg tgg cggacg gag cgc ccg gag gtg aag gcc 537 Gly Pro Glu Gly Ala Val Trp Arg ThrGlu Arg Pro Glu Val Lys Ala 150 155 160 atg gac acc gcc ggc gtc cgc cgcgtg ctc cgg cgc tac gtg tca tcc 585 Met Asp Thr Ala Gly Val Arg Arg ValLeu Arg Arg Tyr Val Ser Ser 165 170 175 gtt gcc gac gag ggg atg gac tgtgcg ctc gcc gcg gcg gtg tac gga 633 Val Ala Asp Glu Gly Met Asp Cys AlaLeu Ala Ala Ala Val Tyr Gly 180 185 190 gga aaa ggt gga gtc ttg tca cagctg cgc gag ctc aag gcg tgg ttc 681 Gly Lys Gly Gly Val Leu Ser Gln LeuArg Glu Leu Lys Ala Trp Phe 195 200 205 210 gag gag cag act ctg ttc cacttc tac tcg gcg tcg att ctt ctg ggc 729 Glu Glu Gln Thr Leu Phe His PheTyr Ser Ala Ser Ile Leu Leu Gly 215 220 225 tat gat gct gct gca gtc gcagca ggc gga gat ggg ggt ggg gtg acg 777 Tyr Asp Ala Ala Ala Val Ala AlaGly Gly Asp Gly Gly Gly Val Thr 230 235 240 gtg aag ctg gtg gac ttt gcccat gtg gcc gag ggt gat ggg gtg att 825 Val Lys Leu Val Asp Phe Ala HisVal Ala Glu Gly Asp Gly Val Ile 245 250 255 gac cac aac ttc ctg ggc gggctc tgc tcg ctg atc aag ttc gtt tct 873 Asp His Asn Phe Leu Gly Gly LeuCys Ser Leu Ile Lys Phe Val Ser 260 265 270 gac att gtt ccg gag act cctcat acg cag cct ttg ggt cct tct taa 921 Asp Ile Val Pro Glu Thr Pro HisThr Gln Pro Leu Gly Pro Ser * 275 280 285 gagaggatcc tggcatttcgatttgataac aaagccctac aagttttgtc tggaaaaaga 981 agcgcctccg agttgtgctgggtgtggaga tctgagacgg tcgtcggccc acttggttgc 1041 cttgcctttg ccttgcctgcaaacatacgg caacctgctc cttttttcgc aaccccttac 1101 ttccgaagaa actttttttttcccactttg ggggttcgat tacgttggat ctggtttgtg 1161 ccactcggtc agaggttgtaagcatggagg gaggcgtgtt gatccggcaa ctgtgtcagt 1221 ctttgcgctg cctgccgtttctgcatggct tttgcctgct gcgatccgat gtgtactgga 1281 gatcgtagtg atggacgtctctacctccaa acgaatccgt ccgataaaaa aaaaaaaaaa 1341 aaa 1344 8 289 PRT Zeamays 8 Met Ser Asp Leu His Pro Pro Glu His Gln Val Ala Gly His Arg Ala 15 10 15 Ser Ala Ser Lys Leu Gly Pro Leu Ile Asp Gly Ser Gly Leu Phe Tyr20 25 30 Lys Pro Leu Gln Ala Gly Asp Arg Gly Glu His Glu Val Ala Phe Tyr35 40 45 Glu Ala Phe Ser Ala His Ala Ala Val Pro Ala Arg Ile Arg Asp Thr50 55 60 Phe Phe Pro Arg Phe His Gly Thr Arg Leu Leu Pro Thr Glu Ala Gln65 70 75 80 Pro Gly Glu Pro His Pro His Leu Val Leu Asp Asp Leu Leu AlaGly 85 90 95 Phe Gln Ala Pro Cys Val Ala Asp Ile Lys Ile Gly Ala Ile ThrTrp 100 105 110 Pro Pro Ser Ser Pro Glu Pro Tyr Ile Ala Lys Cys Leu AlaLys Asp 115 120 125 Arg Gly Thr Thr Ser Val Leu Leu Gly Phe Arg Val SerGly Val Arg 130 135 140 Val Val Gly Pro Glu Gly Ala Val Trp Arg Thr GluArg Pro Glu Val 145 150 155 160 Lys Ala Met Asp Thr Ala Gly Val Arg ArgVal Leu Arg Arg Tyr Val 165 170 175 Ser Ser Val Ala Asp Glu Gly Met AspCys Ala Leu Ala Ala Ala Val 180 185 190 Tyr Gly Gly Lys Gly Gly Val LeuSer Gln Leu Arg Glu Leu Lys Ala 195 200 205 Trp Phe Glu Glu Gln Thr LeuPhe His Phe Tyr Ser Ala Ser Ile Leu 210 215 220 Leu Gly Tyr Asp Ala AlaAla Val Ala Ala Gly Gly Asp Gly Gly Gly 225 230 235 240 Val Thr Val LysLeu Val Asp Phe Ala His Val Ala Glu Gly Asp Gly 245 250 255 Val Ile AspHis Asn Phe Leu Gly Gly Leu Cys Ser Leu Ile Lys Phe 260 265 270 Val SerAsp Ile Val Pro Glu Thr Pro His Thr Gln Pro Leu Gly Pro 275 280 285 Ser9 1105 DNA Glycine max CDS (12)...(851) 9 gcacgagaaa a atg ctc aag atcccg gag cac cag gtg gcc ggg cac aag 50 Met Leu Lys Ile Pro Glu His GlnVal Ala Gly His Lys 1 5 10 gcc aag gac gga atc ctg ggc cca ctc gtc gacgat ttt gga aaa ttc 98 Ala Lys Asp Gly Ile Leu Gly Pro Leu Val Asp AspPhe Gly Lys Phe 15 20 25 tac aag ccc ctc cag acc aac aaa gac gac gac acccgc ggc tcc acc 146 Tyr Lys Pro Leu Gln Thr Asn Lys Asp Asp Asp Thr ArgGly Ser Thr 30 35 40 45 gaa ctc tcc ttt tac acc tct ctc gcc gcc gcc gcccac gac tac tcc 194 Glu Leu Ser Phe Tyr Thr Ser Leu Ala Ala Ala Ala HisAsp Tyr Ser 50 55 60 atc cgc tcc ttc ttc ccc gcc ttt cac ggc acc cgc ctcctg gac gcc 242 Ile Arg Ser Phe Phe Pro Ala Phe His Gly Thr Arg Leu LeuAsp Ala 65 70 75 tcc gac ggc tcc ggt ccc cac cct cac ctg gtc ctg gag gacctc ctc 290 Ser Asp Gly Ser Gly Pro His Pro His Leu Val Leu Glu Asp LeuLeu 80 85 90 tgc ggc tac tcc aaa ccc tcc gtc atg gac gta aag atc ggc tccaga 338 Cys Gly Tyr Ser Lys Pro Ser Val Met Asp Val Lys Ile Gly Ser Arg95 100 105 acc tgg cac ctg gga gac tcc gag gac tac atc tgc aag tgc ctgaag 386 Thr Trp His Leu Gly Asp Ser Glu Asp Tyr Ile Cys Lys Cys Leu Lys110 115 120 125 aag gac aga gag tcc tct agc ttg ccc ttg ggt ttc aga atctcg gga 434 Lys Asp Arg Glu Ser Ser Ser Leu Pro Leu Gly Phe Arg Ile SerGly 130 135 140 gtc aag gac tct atc tcc tcc tgg gaa cct acc agg aaa tctctc cag 482 Val Lys Asp Ser Ile Ser Ser Trp Glu Pro Thr Arg Lys Ser LeuGln 145 150 155 tgt cta tcc gcc cat ggt gtt gca ctt gtt ctc aac aag ttcgtt tcc 530 Cys Leu Ser Ala His Gly Val Ala Leu Val Leu Asn Lys Phe ValSer 160 165 170 tct aat aat atc aac cat gat gat cat cat ccc gat tgc gctttc gca 578 Ser Asn Asn Ile Asn His Asp Asp His His Pro Asp Cys Ala PheAla 175 180 185 acg gag gtc tac ggc gcc gtt ttg gag cgc ttg cag aag ctcaag gac 626 Thr Glu Val Tyr Gly Ala Val Leu Glu Arg Leu Gln Lys Leu LysAsp 190 195 200 205 tgg ttc gag gtt cag acg gtg tat cac ttc tat tct tgttct gtt ctt 674 Trp Phe Glu Val Gln Thr Val Tyr His Phe Tyr Ser Cys SerVal Leu 210 215 220 gtg gtg tac gag aag gat cta ggg aaa ggg aaa gct accaac cct ctg 722 Val Val Tyr Glu Lys Asp Leu Gly Lys Gly Lys Ala Thr AsnPro Leu 225 230 235 gtc aaa ctc gtt gac ttt gca cac gtg gtg gac gga aacggt gtc att 770 Val Lys Leu Val Asp Phe Ala His Val Val Asp Gly Asn GlyVal Ile 240 245 250 gat cac aac ttc ttg ggt ggc ctt tgt tcc ttc atc aagttc ctc aag 818 Asp His Asn Phe Leu Gly Gly Leu Cys Ser Phe Ile Lys PheLeu Lys 255 260 265 gat atc cta gca gta gca tgt ctt cac aag tgactgattttca tcgagttaat 871 Asp Ile Leu Ala Val Ala Cys Leu His Lys * 270275 cttattccta tcagaaaata attatgcttg aattagtgtc gcagactaac tgtttgaagt931 actgtcagaa acaaaataat aatatggact gagaggcaat cttgttctgc taaactccct991 ttcaagttgc tgtcagatac tagccgtccc ttttcctttt tcatattctg tcaaagtgag1051 tcatttaata ataataacaa tgtccttcaa ctccaaaaaa aaaaaaaaaa aaaa 1105 10279 PRT Glycine max 10 Met Leu Lys Ile Pro Glu His Gln Val Ala Gly HisLys Ala Lys Asp 1 5 10 15 Gly Ile Leu Gly Pro Leu Val Asp Asp Phe GlyLys Phe Tyr Lys Pro 20 25 30 Leu Gln Thr Asn Lys Asp Asp Asp Thr Arg GlySer Thr Glu Leu Ser 35 40 45 Phe Tyr Thr Ser Leu Ala Ala Ala Ala His AspTyr Ser Ile Arg Ser 50 55 60 Phe Phe Pro Ala Phe His Gly Thr Arg Leu LeuAsp Ala Ser Asp Gly 65 70 75 80 Ser Gly Pro His Pro His Leu Val Leu GluAsp Leu Leu Cys Gly Tyr 85 90 95 Ser Lys Pro Ser Val Met Asp Val Lys IleGly Ser Arg Thr Trp His 100 105 110 Leu Gly Asp Ser Glu Asp Tyr Ile CysLys Cys Leu Lys Lys Asp Arg 115 120 125 Glu Ser Ser Ser Leu Pro Leu GlyPhe Arg Ile Ser Gly Val Lys Asp 130 135 140 Ser Ile Ser Ser Trp Glu ProThr Arg Lys Ser Leu Gln Cys Leu Ser 145 150 155 160 Ala His Gly Val AlaLeu Val Leu Asn Lys Phe Val Ser Ser Asn Asn 165 170 175 Ile Asn His AspAsp His His Pro Asp Cys Ala Phe Ala Thr Glu Val 180 185 190 Tyr Gly AlaVal Leu Glu Arg Leu Gln Lys Leu Lys Asp Trp Phe Glu 195 200 205 Val GlnThr Val Tyr His Phe Tyr Ser Cys Ser Val Leu Val Val Tyr 210 215 220 GluLys Asp Leu Gly Lys Gly Lys Ala Thr Asn Pro Leu Val Lys Leu 225 230 235240 Val Asp Phe Ala His Val Val Asp Gly Asn Gly Val Ile Asp His Asn 245250 255 Phe Leu Gly Gly Leu Cys Ser Phe Ile Lys Phe Leu Lys Asp Ile Leu260 265 270 Ala Val Ala Cys Leu His Lys 275 11 1195 DNA Eucalyptusgrandis CDS (116)...(1048) 11 gcaccagctt cttggagtag ttgcccatcagcgtggattt tcattttagt ccatctggct 60 gtgatcaatc gaatctgagt aagtttggagaattttttcg cacatcagat acacc atg 118 Met 1 ctc aag gtc ccg gat cat caagtc gcc ggt cac cgg gga gac ggg gga 166 Leu Lys Val Pro Asp His Gln ValAla Gly His Arg Gly Asp Gly Gly 5 10 15 aag ctg ggg cca ctg gtg gat gattcg ggc cgc ttc tat aag cct ctc 214 Lys Leu Gly Pro Leu Val Asp Asp SerGly Arg Phe Tyr Lys Pro Leu 20 25 30 cag agc gat cat cgc gga gac acg gaagtg gcc ttt tac gag tca ttc 262 Gln Ser Asp His Arg Gly Asp Thr Glu ValAla Phe Tyr Glu Ser Phe 35 40 45 tat tcc aat acc gag atc cca ggt cac attcgc aaa ttc ttt cct gcg 310 Tyr Ser Asn Thr Glu Ile Pro Gly His Ile ArgLys Phe Phe Pro Ala 50 55 60 65 ttt cac gga act aag act att gag gcg tctgat gga tcg ggt cct caa 358 Phe His Gly Thr Lys Thr Ile Glu Ala Ser AspGly Ser Gly Pro Gln 70 75 80 cct cac ctg gtt ctg gag gat ctc gtc tcg ggtcgc acg aac cca tct 406 Pro His Leu Val Leu Glu Asp Leu Val Ser Gly ArgThr Asn Pro Ser 85 90 95 ctc atg gac atc aag act gga tcc aga aca tgg tatccg gag gcc tct 454 Leu Met Asp Ile Lys Thr Gly Ser Arg Thr Trp Tyr ProGlu Ala Ser 100 105 110 gag gag tac atc caa aag tgc tta gag aaa gat cgaaat agc aca agc 502 Glu Glu Tyr Ile Gln Lys Cys Leu Glu Lys Asp Arg AsnSer Thr Ser 115 120 125 gtt tca ttg ggt ttt agg att tct ggg cta agg gtatat caa aat agc 550 Val Ser Leu Gly Phe Arg Ile Ser Gly Leu Arg Val TyrGln Asn Ser 130 135 140 145 gaa gct gga ttt tgg caa cct gag aag aag gttgtt tat agc ttt aat 598 Glu Ala Gly Phe Trp Gln Pro Glu Lys Lys Val ValTyr Ser Phe Asn 150 155 160 gcg gac ggt gtc agg tcg gct ctg agg aag tttgtt tct tcc aac ttg 646 Ala Asp Gly Val Arg Ser Ala Leu Arg Lys Phe ValSer Ser Asn Leu 165 170 175 tct ctg ggt cca aat gtg gat ccg gat tgt ttgtat gca tca aaa gtt 694 Ser Leu Gly Pro Asn Val Asp Pro Asp Cys Leu TyrAla Ser Lys Val 180 185 190 tac tgt cac cgg ggt gga att ttg gca caa ttgctt cag ctg aag gaa 742 Tyr Cys His Arg Gly Gly Ile Leu Ala Gln Leu LeuGln Leu Lys Glu 195 200 205 tgg ttt gag gtt cag acg aat tat cac ttc tattct tgt tca ctc att 790 Trp Phe Glu Val Gln Thr Asn Tyr His Phe Tyr SerCys Ser Leu Ile 210 215 220 225 atc tta tat gac agg gag tct gct ttg gacggc tgt gca cac ccg aaa 838 Ile Leu Tyr Asp Arg Glu Ser Ala Leu Asp GlyCys Ala His Pro Lys 230 235 240 gtt aaa ctg gtg gac ttt gca cat gtg atggat ggc cac ggc gtg atc 886 Val Lys Leu Val Asp Phe Ala His Val Met AspGly His Gly Val Ile 245 250 255 gat cac aac ttc ttg ggt ggc ctc tgt tctgta atc aag ttt ata cgt 934 Asp His Asn Phe Leu Gly Gly Leu Cys Ser ValIle Lys Phe Ile Arg 260 265 270 gac att gct gat gaa gat aac aag tgt gcaaag tgc gaa gtc aat ctt 982 Asp Ile Ala Asp Glu Asp Asn Lys Cys Ala LysCys Glu Val Asn Leu 275 280 285 gga ttg aaa gaa aat ggc ttc tat aag agcagc acg gaa cca gag ctt 1030 Gly Leu Lys Glu Asn Gly Phe Tyr Lys Ser SerThr Glu Pro Glu Leu 290 295 300 305 gat cac gag gcc tgc tag tggaaactggagaataactg cattcatgca 1078 Asp His Glu Ala Cys * 310 ttcctgcattcctgctctga caagtggttc agaatgggta taataacagt ctattttagt 1138 caaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaa 1195 12 310 PRTEucalyptus grandis 12 Met Leu Lys Val Pro Asp His Gln Val Ala Gly HisArg Gly Asp Gly 1 5 10 15 Gly Lys Leu Gly Pro Leu Val Asp Asp Ser GlyArg Phe Tyr Lys Pro 20 25 30 Leu Gln Ser Asp His Arg Gly Asp Thr Glu ValAla Phe Tyr Glu Ser 35 40 45 Phe Tyr Ser Asn Thr Glu Ile Pro Gly His IleArg Lys Phe Phe Pro 50 55 60 Ala Phe His Gly Thr Lys Thr Ile Glu Ala SerAsp Gly Ser Gly Pro 65 70 75 80 Gln Pro His Leu Val Leu Glu Asp Leu ValSer Gly Arg Thr Asn Pro 85 90 95 Ser Leu Met Asp Ile Lys Thr Gly Ser ArgThr Trp Tyr Pro Glu Ala 100 105 110 Ser Glu Glu Tyr Ile Gln Lys Cys LeuGlu Lys Asp Arg Asn Ser Thr 115 120 125 Ser Val Ser Leu Gly Phe Arg IleSer Gly Leu Arg Val Tyr Gln Asn 130 135 140 Ser Glu Ala Gly Phe Trp GlnPro Glu Lys Lys Val Val Tyr Ser Phe 145 150 155 160 Asn Ala Asp Gly ValArg Ser Ala Leu Arg Lys Phe Val Ser Ser Asn 165 170 175 Leu Ser Leu GlyPro Asn Val Asp Pro Asp Cys Leu Tyr Ala Ser Lys 180 185 190 Val Tyr CysHis Arg Gly Gly Ile Leu Ala Gln Leu Leu Gln Leu Lys 195 200 205 Glu TrpPhe Glu Val Gln Thr Asn Tyr His Phe Tyr Ser Cys Ser Leu 210 215 220 IleIle Leu Tyr Asp Arg Glu Ser Ala Leu Asp Gly Cys Ala His Pro 225 230 235240 Lys Val Lys Leu Val Asp Phe Ala His Val Met Asp Gly His Gly Val 245250 255 Ile Asp His Asn Phe Leu Gly Gly Leu Cys Ser Val Ile Lys Phe Ile260 265 270 Arg Asp Ile Ala Asp Glu Asp Asn Lys Cys Ala Lys Cys Glu ValAsn 275 280 285 Leu Gly Leu Lys Glu Asn Gly Phe Tyr Lys Ser Ser Thr GluPro Glu 290 295 300 Leu Asp His Glu Ala Cys 305 310 13 1020 DNAParthenium argentatum CDS (21)...(908) 13 gcacgagaac ttcttcagac atg ctcaag gcc cca gat cat cag gtt gct gga 53 Met Leu Lys Ala Pro Asp His GlnVal Ala Gly 1 5 10 cat gaa gct ggg ctc ggg aag ctt ggc cca ctc att gatgat tca ggc 101 His Glu Ala Gly Leu Gly Lys Leu Gly Pro Leu Ile Asp AspSer Gly 15 20 25 cgg ttt tac aaa cca ctg cag ggt gat aac cgt ggg tca gaagaa gta 149 Arg Phe Tyr Lys Pro Leu Gln Gly Asp Asn Arg Gly Ser Glu GluVal 30 35 40 gcc ttt tat gaa tca ttt tct tct aac aat aat att cca gaa cacata 197 Ala Phe Tyr Glu Ser Phe Ser Ser Asn Asn Asn Ile Pro Glu His Ile45 50 55 cgc aaa ttc ttt cct ata tat tat ggc acc aaa atc atg aag gca tcc245 Arg Lys Phe Phe Pro Ile Tyr Tyr Gly Thr Lys Ile Met Lys Ala Ser 6065 70 75 act ggc tct gac cat cct cac atg gtg ttg caa gat ctt aca tca gct293 Thr Gly Ser Asp His Pro His Met Val Leu Gln Asp Leu Thr Ser Ala 8085 90 cat gtc aac cca tct gta atg gac atc aaa atc ggg tcc aga aca tgg341 His Val Asn Pro Ser Val Met Asp Ile Lys Ile Gly Ser Arg Thr Trp 95100 105 gcg cca gaa gct tcc gag gcg tac att gca aaa tgc tta aaa aag gat389 Ala Pro Glu Ala Ser Glu Ala Tyr Ile Ala Lys Cys Leu Lys Lys Asp 110115 120 agg gaa agc aca agt att cca ttg gga ttc agg atc tcc ggg ctg caa437 Arg Glu Ser Thr Ser Ile Pro Leu Gly Phe Arg Ile Ser Gly Leu Gln 125130 135 gtc tat atc gat gat ggg tca ggg ttt tat aag cct cat aga aat tac485 Val Tyr Ile Asp Asp Gly Ser Gly Phe Tyr Lys Pro His Arg Asn Tyr 140145 150 155 atg cgt aaa acc ggc cca gct gat gtt aga cta ctt ctt agg aaattt 533 Met Arg Lys Thr Gly Pro Ala Asp Val Arg Leu Leu Leu Arg Lys Phe160 165 170 gtt tct tct aac ccg tct gca gag atg gaa atg cgc aca ggc ctaggc 581 Val Ser Ser Asn Pro Ser Ala Glu Met Glu Met Arg Thr Gly Leu Gly175 180 185 ccg gat tgt tct tta gca tct ttt gtt tat ggt ggg cct aat gggata 629 Pro Asp Cys Ser Leu Ala Ser Phe Val Tyr Gly Gly Pro Asn Gly Ile190 195 200 tta gct caa ctg atg gaa ttg aag aca tgg ttt gaa gat caa acaatt 677 Leu Ala Gln Leu Met Glu Leu Lys Thr Trp Phe Glu Asp Gln Thr Ile205 210 215 tac cac ttc tat gct tgt tct ttt ttg ttc atc ttt gaa aag aggttg 725 Tyr His Phe Tyr Ala Cys Ser Phe Leu Phe Ile Phe Glu Lys Arg Leu220 225 230 235 gtg tta aaa ggt gct cgg tca aac gca gaa gtc aaa ctt attgat ttt 773 Val Leu Lys Gly Ala Arg Ser Asn Ala Glu Val Lys Leu Ile AspPhe 240 245 250 gct cat gtt aca gat ggt aat ggt gtt att gat cac aat ttcttg ggt 821 Ala His Val Thr Asp Gly Asn Gly Val Ile Asp His Asn Phe LeuGly 255 260 265 ggg ctc tgt tct ttg ata aag ttc att tct gac ata ctt tcggag aca 869 Gly Leu Cys Ser Leu Ile Lys Phe Ile Ser Asp Ile Leu Ser GluThr 270 275 280 aaa gat tgt aat ggt aca aac ggt cag gtt gaa ctt tgaaactctcttc 918 Lys Asp Cys Asn Gly Thr Asn Gly Gln Val Glu Leu * 285 290295 ttgttgcttt tcttcaataa tttatcatga cagtgtttaa ttgtaaagat attcgcttac978 cggaatatat cttggttatg agtgaaaaaa aaaaaaaaaa aa 1020 14 295 PRTParthenium argentatum 14 Met Leu Lys Ala Pro Asp His Gln Val Ala Gly HisGlu Ala Gly Leu 1 5 10 15 Gly Lys Leu Gly Pro Leu Ile Asp Asp Ser GlyArg Phe Tyr Lys Pro 20 25 30 Leu Gln Gly Asp Asn Arg Gly Ser Glu Glu ValAla Phe Tyr Glu Ser 35 40 45 Phe Ser Ser Asn Asn Asn Ile Pro Glu His IleArg Lys Phe Phe Pro 50 55 60 Ile Tyr Tyr Gly Thr Lys Ile Met Lys Ala SerThr Gly Ser Asp His 65 70 75 80 Pro His Met Val Leu Gln Asp Leu Thr SerAla His Val Asn Pro Ser 85 90 95 Val Met Asp Ile Lys Ile Gly Ser Arg ThrTrp Ala Pro Glu Ala Ser 100 105 110 Glu Ala Tyr Ile Ala Lys Cys Leu LysLys Asp Arg Glu Ser Thr Ser 115 120 125 Ile Pro Leu Gly Phe Arg Ile SerGly Leu Gln Val Tyr Ile Asp Asp 130 135 140 Gly Ser Gly Phe Tyr Lys ProHis Arg Asn Tyr Met Arg Lys Thr Gly 145 150 155 160 Pro Ala Asp Val ArgLeu Leu Leu Arg Lys Phe Val Ser Ser Asn Pro 165 170 175 Ser Ala Glu MetGlu Met Arg Thr Gly Leu Gly Pro Asp Cys Ser Leu 180 185 190 Ala Ser PheVal Tyr Gly Gly Pro Asn Gly Ile Leu Ala Gln Leu Met 195 200 205 Glu LeuLys Thr Trp Phe Glu Asp Gln Thr Ile Tyr His Phe Tyr Ala 210 215 220 CysSer Phe Leu Phe Ile Phe Glu Lys Arg Leu Val Leu Lys Gly Ala 225 230 235240 Arg Ser Asn Ala Glu Val Lys Leu Ile Asp Phe Ala His Val Thr Asp 245250 255 Gly Asn Gly Val Ile Asp His Asn Phe Leu Gly Gly Leu Cys Ser Leu260 265 270 Ile Lys Phe Ile Ser Asp Ile Leu Ser Glu Thr Lys Asp Cys AsnGly 275 280 285 Thr Asn Gly Gln Val Glu Leu 290 295 15 899 DNA Zea maysCDS (89)...(424) 15 gccccaaaat ctctttctcc gctgcgccgc aaacccaccgcttccaccat cgccacccgt 60 caccccttgc tcccatagtc cccatacc atg ccc gac ctccac ccg ccg gag 112 Met Pro Asp Leu His Pro Pro Glu 1 5 cac caa gtc gccggt cac cgc gcc tcc gcc agc aag ctg ggc cca ctc 160 His Gln Val Ala GlyHis Arg Ala Ser Ala Ser Lys Leu Gly Pro Leu 10 15 20 atc gac ggc tct ggcctc ttc tac aag ccg ctc cag gcc ggc gac cgt 208 Ile Asp Gly Ser Gly LeuPhe Tyr Lys Pro Leu Gln Ala Gly Asp Arg 25 30 35 40 ggg gag cac gag gtcgcc ttc tat gag gcg ttc tcc gcc cac gcc gcc 256 Gly Glu His Glu Val AlaPhe Tyr Glu Ala Phe Ser Ala His Ala Ala 45 50 55 gtc ccg gcc cgc atc cgagac acc ttc ttc ccc cgg ttc cac ggc acg 304 Val Pro Ala Arg Ile Arg AspThr Phe Phe Pro Arg Phe His Gly Thr 60 65 70 cga ctc ctc ccc acc gag gcgcag ccc ggg gag ccg cat ccg tac ctc 352 Arg Leu Leu Pro Thr Glu Ala GlnPro Gly Glu Pro His Pro Tyr Leu 75 80 85 gtc ctc gac gac ctc ctc gcg gggttt gag gcg ccc tgc gtc gca gac 400 Val Leu Asp Asp Leu Leu Ala Gly PheGlu Ala Pro Cys Val Ala Asp 90 95 100 atc aag atc ggt gcc atc acg tgaccatgagcga tctgctcgga ttccacgtct 454 Ile Lys Ile Gly Ala Ile Thr * 105110 ccggcgtccg agtcgtcggc cccgagggcg ccgtgtggcg gacggagcgc cctgaggtga514 aggctatgga cattgtcggc gtccgccgcg tgctccggcg ctgcatgtca tccgcttgcc574 ggcgagggga tggactgcgc gctcgcggcg gcggtgtacg gaggaaaagg tggagtcttg634 tcacagctgc gcgagctcaa ggcgtggttc gaggggcaga ctctgttcca cttctactcg694 gcgtcgattc ttctgggcta tgatgctgct gcagtcgcag caggcggagg tgggggtggg754 gtaacagtga agctggtgga ccttgcccat gtggccgagg gtgatggggt gattgaccac814 aacttcctgg gcgggctctg ctagctgatc aagtttgttt ctgacattgt tccagagact874 ccttagacgc agcaagggcg aattc 899 16 111 PRT Zea mays 16 Met Pro AspLeu His Pro Pro Glu His Gln Val Ala Gly His Arg Ala 1 5 10 15 Ser AlaSer Lys Leu Gly Pro Leu Ile Asp Gly Ser Gly Leu Phe Tyr 20 25 30 Lys ProLeu Gln Ala Gly Asp Arg Gly Glu His Glu Val Ala Phe Tyr 35 40 45 Glu AlaPhe Ser Ala His Ala Ala Val Pro Ala Arg Ile Arg Asp Thr 50 55 60 Phe PhePro Arg Phe His Gly Thr Arg Leu Leu Pro Thr Glu Ala Gln 65 70 75 80 ProGly Glu Pro His Pro Tyr Leu Val Leu Asp Asp Leu Leu Ala Gly 85 90 95 PheGlu Ala Pro Cys Val Ala Asp Ile Lys Ile Gly Ala Ile Thr 100 105 110 17643 DNA Zea mays misc_feature (1)...(643) n = A, T, C or G 17 ggccgtccctgnttttgtta accaccccgc cccaaaatct ctttctccgc tgcgctgcaa 60 acccaccgcttccaccatcg ccactcgtca ccccttgctc ccatagtccc cataccatgc 120 ccgacctccacccgccggag caccaagtcg ccggtcaccg cgcctccgcc agcaagctgg 180 gcccgctcatcgacggctcc ggcctcttct acaagccgct ccaggccggc gaccgtgggg 240 agcacgaggtcgccttctat gaggcgttct ccgcccacgc cgncgtcccg gcccgcatcc 300 gagacaccttcttcccccgg ttccacggca cgcgactcct ccccaccgag gcgcagcccg 360 gggagccgcatccgcacctc gtcctcgacg acctcctcgc ggggtttgag gcgccctgcg 420 tcgcagacatcaagatcggc gccatcacgt ggccaccgag ttcgccggag ccctacatcg 480 ncaagtacctngccaaggac cgcgggacca cgagcgttct gctcggattc cgcgtcttgc 540 gtccgagtcgtcggccccga gggcgccgtg tggcggacgg agcgccccgg gggtgaangc 600 tatggacacccgtcggngnc cggcgngtgc ttcgggngct acg 643 18 519 DNA Zea maysmisc_feature (1)...(519) n = A, T, C, or G 18 ggtacggang aaaangtggagtcttgtcac agctgcgcga gctcaangcg tggttcgagg 60 ggcagactct gttccacttctactcggcgt cgattcttct gggctatgat gctgctgcag 120 tcgcagcagg cggangtgggggtggggtaa cagtgaagct ggtggacttt gcccatgtgg 180 ccgagggtga tggggtgattgaccacaact tcctgggcgg gctctgctan ctgatcaagt 240 ttgtttctga cattgttccagagactcctc agacgcagcc tttgggtcct tcttaagaaa 300 agatcctggc attttcgatttgataacaaa ggaancactt tcagctgcca aaaaaaaanc 360 accagtgaag atgaaaataacattattgag gaaagttccg atnataaccc accanattna 420 aaaaaaaaag gtcccaaatttccgaaaatn tggatcttaa gaataatctc ctgaaaacan 480 aattataaaa cgtgaaaaccccggctncnt catttacnc 519 19 353 DNA Zea mays misc_feature (1)...(353) n= A, T, C, or G 19 ctcaaggcat ggttggagga gcagactctg ttccacttctactcggcgtc gattcttctg 60 ggctatgatg ctgctgcagt cgcancaggc ggaggtgggggtggggtaac agtgaagctg 120 gtggactttg cccatgtggc cgagggtgat ggggttgatttgaccacaac ttcctgggcg 180 agctctgcta gctgatcaag ttccgtttct tgacattgttccaganactc cttagacgcc 240 agcctttggg tccttcctta aaaaaagatc cctgacntttttgatttgat tacnaaggaa 300 acactttcca cttgccnaaa aaaaaagccc ntgaggattaaaaaattaac ntt 353 20 3416 DNA Zea mays CDS (72)...(407) 20 ccacgcgtccggcaaaccca ccgcttccac catcgccacc cgtcacccct tgctcccata 60 gtccccatac catg ccc gac ctc cac ccg ccg gag cac caa gtc gcc ggt 110 Met Pro Asp LeuHis Pro Pro Glu His Gln Val Ala Gly 1 5 10 cac cgc gcc tcc gcc agc aagctg ggc cca ctc atc gac gac tct ggc 158 His Arg Ala Ser Ala Ser Lys LeuGly Pro Leu Ile Asp Asp Ser Gly 15 20 25 ctc ttc tac aag ccg ctc cag gccggc gac cgt ggg gag cac gag gtc 206 Leu Phe Tyr Lys Pro Leu Gln Ala GlyAsp Arg Gly Glu His Glu Val 30 35 40 45 gcc ttc tat gag gcg ttc tcc gcccac gcc gcc gtc ccg gcc cgc atc 254 Ala Phe Tyr Glu Ala Phe Ser Ala HisAla Ala Val Pro Ala Arg Ile 50 55 60 cga gac acc ttc ttc ccc cgg ttc cacggc acg cga ctc ctc ccc acc 302 Arg Asp Thr Phe Phe Pro Arg Phe His GlyThr Arg Leu Leu Pro Thr 65 70 75 gag gcg cag ccc ggg gag ccg cat ccg cacctc gtc ctc gac gac ctc 350 Glu Ala Gln Pro Gly Glu Pro His Pro His LeuVal Leu Asp Asp Leu 80 85 90 ctc gcg ggg ttt gag gcg ccc tgc gtc gca gacatc aag atc ggt gcc 398 Leu Ala Gly Phe Glu Ala Pro Cys Val Ala Asp IleLys Ile Gly Ala 95 100 105 atc acg tga ccacgagcgt tctgctcgga ttccgcgtctccggcgtccg 447 Ile Thr * 110 agtcgtcggc cccgagggcg ccgtgtggcg gacggagcgcccggaggtga aggctatgga 507 cattgtcggc gtccgccgcg tgctccggcg ctacgtgtcatccgcttgcc gacgagggga 567 tggactgcgc gctcgcggcg gcggtgtacg gaggaaaaggtggagtcttg tcacagctgc 627 gcgagctcaa ggcgtggttc gaggggcaga ctctgttccacttctactcg gcgtcgattc 687 ttctgggcta tgatgctgct gcagtcgcag caggcggaggtgggggtggg gtaacagtga 747 agctggtgga ctttgcccat gtggccgagg gtgatggggtgattgaccac aacttcctgg 807 gcgggctctg ctagctgatc aagtttgttt ctgacattgttccagagact cctcagacgc 867 agcctttggg tccttcttaa gagaggatcc tggcattttcgatttgataa caaaggaagc 927 actttcagct gcaaaaaaag aaagcagcag tgaggatgaagatgacagta gtgaggaaag 987 ttcggatgat gagccaacaa aagttgaaga aaagaaggctccaaaagtat cagaaaatat 1047 tggatctgag gatgaatctt ctgaagacaa gagtgataaagacagtgaag agcctcaggc 1107 atgccatcat ttaacacctc aggcatgcca tcatttttgtttcacaactc aaaagtaaag 1167 gaaaacagta aaagtatgca ggcagtatga gggacacacatagtttactg aaactccctt 1227 acacagacac atacacaccg tgttcactga aacattcagatttcactaaa ctgcaacttc 1287 tccaaacaaa cactatctgc ggctcggtca agtaacgagcctcggctcgg ctcgctcctc 1347 tagcgagcct aaaaagtcgg ctcggttcgg cgagccaacgagcctgacca taagcatgaa 1407 atcagtctcc aaaatataat ataaagtctc aaaaataatttaagtgacac gtcttaaatt 1467 agtaaaataa atatatatca tataatatag aaaataagttaattttgtac agtaatctaa 1527 aaaatataaa ttaatcatct atttagtacc tataatatatgttaattaaa atttatataa 1587 caaaaatgtt gttgtttgag ccagctcgcg agctgaactggctcgctctg gctcgctctt 1647 ttattgagcc agaaaaaact ctgctcgagc ttgttctaagcacagtttct ggatcggagg 1707 agcatccccg cctaggtctc tgcagccatg gttcgcggatcgctcggcaa gcttgcatcg 1767 cgcgccctct ccgtcgccgg gagatggcag caccagcagctccgccgcct caacatccac 1827 gagtaccagg gcgcggagtt gatgggtaaa tacgggatcaacgtgcccag gggcgcggcg 1887 gctgggtccg tacatgaggt caaggacgcc ttgaagaacatgttccccag cgagaaagag 1947 atagttgtta aaagtcaaat ccttgctggt ggccgagggctgggaacttt caaaagcgga 2007 ctgcaaggtg gtgtccatat tgttaaggct gaggaagctgaattgattgc aagtaaaatg 2067 ttaggccaga ttctgataac gaaacaaact ggtccagagggaaagattgt gagcaaggtc 2127 tacttgtgtg agaaactatc tcttactaat gagatgtactttgccatcac ccttgatagg 2187 aaaactgctg gtccgctcat tattgcttgc agcaagggaggaaaacacta tagttgacct 2247 caatgttcaa aggatggcca gggctacatc atcttgttgttgacgggttc cgtgtgttca 2307 atcgccgagc agaaagccag gaacagaact taggcgttggcgattggcat ctccctcccc 2367 taagccatgg ccaccgggcg gcccgtacga ctcgtgctggatgcctccct cctcctcgac 2427 ccctcctcca ccagggaggc ggcggcggtg gctctgcggcccggggtaga ggagctgttg 2487 cggcggttgc gctactccaa cctgaatgtg gcaatctgctatgcagaggg catgccaact 2547 aatgagatgc tctacttatc tacattatta ttacatccctctgaagttgt atcttcagaa 2607 gttcacattg acagtatttg cttcctcttg ccatacttacccatcatggc ccatggggtg 2667 tctatcttat catgccatct tcaaagaatg gcatcatgttaacaaaaatg aatgagaaat 2727 cagtcatttc taatggaaag tcaggctttc ttgaaaaggtcgcaagctca cacttgtttg 2787 gctctatagc acttcttgcg aaaagtggga atctttctctaactgaatta atgttagaat 2847 ggagccaaac aagtttatgt ttttatgcga cttcaagagttgacaaaggt ttaagttctg 2907 agctccagaa tcagaattgg agagttcttt ctgtagctaatgaatgtagc atagaggttc 2967 ctggtgtttt aaatgttcaa aggcttcagc agttgcttctcaccttggct actctaataa 3027 aagggaacta tgtgactcat ctgttctggt gattggatatataatgaaaa tattctgtga 3087 ggaagacttc gcaaggagat gtggttctgt cacttatgtgaccgttgtcg tgtatggaga 3147 cgtgtatgga gacgaggaca agccagcgct tataatgtttacagagatgt ggttctgtga 3207 ctgttgccgt gtactcaggc tttatttcaa caagatttaaatatgagatg tagagtgatt 3267 gatgtacatc acttcactaa tcatgaaatc tgtagaaggcgaaactacta gccatatatg 3327 atatgcataa tccgtgtggt aaacattatc aatatcacacaaattatttc taatgggttt 3387 tgaattatca aaaaaaaaaa aaaaaaaaa 3416 21 111PRT Zea mays 21 Met Pro Asp Leu His Pro Pro Glu His Gln Val Ala Gly HisArg Ala 1 5 10 15 Ser Ala Ser Lys Leu Gly Pro Leu Ile Asp Asp Ser GlyLeu Phe Tyr 20 25 30 Lys Pro Leu Gln Ala Gly Asp Arg Gly Glu His Glu ValAla Phe Tyr 35 40 45 Glu Ala Phe Ser Ala His Ala Ala Val Pro Ala Arg IleArg Asp Thr 50 55 60 Phe Phe Pro Arg Phe His Gly Thr Arg Leu Leu Pro ThrGlu Ala Gln 65 70 75 80 Pro Gly Glu Pro His Pro His Leu Val Leu Asp AspLeu Leu Ala Gly 85 90 95 Phe Glu Ala Pro Cys Val Ala Asp Ile Lys Ile GlyAla Ile Thr 100 105 110 22 1448 DNA Parthenium argentatum CDS(52)...(1020) 22 gcacgaggca cactcaatgg ctccgatgct cagaggccaa cggagggtacc atg ctg 57 Met Leu 1 cca gct cca gct gtt cct aat ggc acg ggt gct ccgctt aag gac gaa 105 Pro Ala Pro Ala Val Pro Asn Gly Thr Gly Ala Pro LeuLys Asp Glu 5 10 15 cct tcc aac ccc gat cag gcg cag cac cag cct gac gagcgc gtt caa 153 Pro Ser Asn Pro Asp Gln Ala Gln His Gln Pro Asp Glu ArgVal Gln 20 25 30 cac ttc atc ctt ctt gaa gac ctt act gct ggc atg aca aggcct tgt 201 His Phe Ile Leu Leu Glu Asp Leu Thr Ala Gly Met Thr Arg ProCys 35 40 45 50 gtc tta gac ttg aag atg ggt acg cgc cag tat ggt gtg gaagcc gat 249 Val Leu Asp Leu Lys Met Gly Thr Arg Gln Tyr Gly Val Glu AlaAsp 55 60 65 gag aag aaa cag cgg tct caa cgg cgc aag tgt cag atg acc accagt 297 Glu Lys Lys Gln Arg Ser Gln Arg Arg Lys Cys Gln Met Thr Thr Ser70 75 80 gct caa ctc ggc gtg cga gtc tgc ggt atg caa att tgg aac gcc aag345 Ala Gln Leu Gly Val Arg Val Cys Gly Met Gln Ile Trp Asn Ala Lys 8590 95 acc cag agc tac atc ttc gag gac aag tac ttc ggt cga gat ctg aaa393 Thr Gln Ser Tyr Ile Phe Glu Asp Lys Tyr Phe Gly Arg Asp Leu Lys 100105 110 gca gga aaa gaa ttt cag gac gcg ctt aag cgc ttt ttt tgg gat ggg441 Ala Gly Lys Glu Phe Gln Asp Ala Leu Lys Arg Phe Phe Trp Asp Gly 115120 125 130 acg agc tac aaa gca gca aac aga cac ata ccc gtc ata ttg gagaag 489 Thr Ser Tyr Lys Ala Ala Asn Arg His Ile Pro Val Ile Leu Glu Lys135 140 145 atc agc caa ctg gaa cgc atg ata cga aaa ctt cca gga tac agattc 537 Ile Ser Gln Leu Glu Arg Met Ile Arg Lys Leu Pro Gly Tyr Arg Phe150 155 160 tac gcg agt agt ttg ttg atg ctc tat gat cgt ggg gac ggt gagtcg 585 Tyr Ala Ser Ser Leu Leu Met Leu Tyr Asp Arg Gly Asp Gly Glu Ser165 170 175 aag gag aaa gac gca gca ccc tct tta cct aac ggc ttg tcg aacccg 633 Lys Glu Lys Asp Ala Ala Pro Ser Leu Pro Asn Gly Leu Ser Asn Pro180 185 190 agc aac gaa gac gtt tca aca ata cca tct gga ctt aca tca ccaggg 681 Ser Asn Glu Asp Val Ser Thr Ile Pro Ser Gly Leu Thr Ser Pro Gly195 200 205 210 ccg aca gtc gct tct aaa ccg tca ccc aag aag cac gga gagatc aag 729 Pro Thr Val Ala Ser Lys Pro Ser Pro Lys Lys His Gly Glu IleLys 215 220 225 ctg aaa att gtc gac ttt gcc aac tgc gtg act gca gaa gaccct cta 777 Leu Lys Ile Val Asp Phe Ala Asn Cys Val Thr Ala Glu Asp ProLeu 230 235 240 cca gac gac tta cct tgt cca cct gaa aat ccc gac ggc atcgat aga 825 Pro Asp Asp Leu Pro Cys Pro Pro Glu Asn Pro Asp Gly Ile AspArg 245 250 255 ggg tac ctc cgt ggc ctc cga tca cta cgc ctc tac ttc caacgc att 873 Gly Tyr Leu Arg Gly Leu Arg Ser Leu Arg Leu Tyr Phe Gln ArgIle 260 265 270 tgg aat gac atc aac gag gaa tgg gtc gaa cga ggc gag ggcgag ggc 921 Trp Asn Asp Ile Asn Glu Glu Trp Val Glu Arg Gly Glu Gly GluGly 275 280 285 290 atg gcg cga aat cat cac cat ggc cct ggt tta ggt gaggtt ggt gcg 969 Met Ala Arg Asn His His His Gly Pro Gly Leu Gly Glu ValGly Ala 295 300 305 ggc tgg atg gat gat gct ggt ggt gag gat aca ggc tacgcc agt ttc 1017 Gly Trp Met Asp Asp Ala Gly Gly Glu Asp Thr Gly Tyr AlaSer Phe 310 315 320 taa agaagaggag gaacagcaaa gctgcccacg ctcgacagaagtcggacagt 1070 cgatattgat acgtccatcc cttttccctt cccttcattt ccacgttcagtctatttcac 1130 attgtgtgca ttttgggttg caagcatggt gttttggtgc ataatggtaagacaaagggt 1190 aatgaaattg gcaactcttt tggcatgcat cggcgcagca ttttatgggcggtcagaacc 1250 tctgcgttgt ggcttttagt ttttgaaatt tgcggaatct ggggtgttcttgaggcggat 1310 tctttgtata ttatcataaa gagtagggta gcgctagctc attaatacaacactttgaat 1370 gtcgtcaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa 1430 aaaaaaaaaa aaaaaaaa 1448 23 322 PRT Partheniumargentatum 23 Met Leu Pro Ala Pro Ala Val Pro Asn Gly Thr Gly Ala ProLeu Lys 1 5 10 15 Asp Glu Pro Ser Asn Pro Asp Gln Ala Gln His Gln ProAsp Glu Arg 20 25 30 Val Gln His Phe Ile Leu Leu Glu Asp Leu Thr Ala GlyMet Thr Arg 35 40 45 Pro Cys Val Leu Asp Leu Lys Met Gly Thr Arg Gln TyrGly Val Glu 50 55 60 Ala Asp Glu Lys Lys Gln Arg Ser Gln Arg Arg Lys CysGln Met Thr 65 70 75 80 Thr Ser Ala Gln Leu Gly Val Arg Val Cys Gly MetGln Ile Trp Asn 85 90 95 Ala Lys Thr Gln Ser Tyr Ile Phe Glu Asp Lys TyrPhe Gly Arg Asp 100 105 110 Leu Lys Ala Gly Lys Glu Phe Gln Asp Ala LeuLys Arg Phe Phe Trp 115 120 125 Asp Gly Thr Ser Tyr Lys Ala Ala Asn ArgHis Ile Pro Val Ile Leu 130 135 140 Glu Lys Ile Ser Gln Leu Glu Arg MetIle Arg Lys Leu Pro Gly Tyr 145 150 155 160 Arg Phe Tyr Ala Ser Ser LeuLeu Met Leu Tyr Asp Arg Gly Asp Gly 165 170 175 Glu Ser Lys Glu Lys AspAla Ala Pro Ser Leu Pro Asn Gly Leu Ser 180 185 190 Asn Pro Ser Asn GluAsp Val Ser Thr Ile Pro Ser Gly Leu Thr Ser 195 200 205 Pro Gly Pro ThrVal Ala Ser Lys Pro Ser Pro Lys Lys His Gly Glu 210 215 220 Ile Lys LeuLys Ile Val Asp Phe Ala Asn Cys Val Thr Ala Glu Asp 225 230 235 240 ProLeu Pro Asp Asp Leu Pro Cys Pro Pro Glu Asn Pro Asp Gly Ile 245 250 255Asp Arg Gly Tyr Leu Arg Gly Leu Arg Ser Leu Arg Leu Tyr Phe Gln 260 265270 Arg Ile Trp Asn Asp Ile Asn Glu Glu Trp Val Glu Arg Gly Glu Gly 275280 285 Glu Gly Met Ala Arg Asn His His His Gly Pro Gly Leu Gly Glu Val290 295 300 Gly Ala Gly Trp Met Asp Asp Ala Gly Gly Glu Asp Thr Gly TyrAla 305 310 315 320 Ser Phe 24 2270 DNA Zea mays CDS (3)...(953) 24 ccacg cgt ccg cga aaa ttg aga aac att gtt cag tgg acg ccg ttc 47 Thr ArgPro Arg Lys Leu Arg Asn Ile Val Gln Trp Thr Pro Phe 1 5 10 15 ttt caaact tac aaa aaa cag agg tat cca tgg gta cag cta gcc gga 95 Phe Gln ThrTyr Lys Lys Gln Arg Tyr Pro Trp Val Gln Leu Ala Gly 20 25 30 cac caa ggcaat ttc aaa gcc ggt ccg gaa cct ggt acg atc ctc aag 143 His Gln Gly AsnPhe Lys Ala Gly Pro Glu Pro Gly Thr Ile Leu Lys 35 40 45 aaa ctt tgt cccaaa gaa cag ttg tgc ttc caa gtg ctg atg aag gac 191 Lys Leu Cys Pro LysGlu Gln Leu Cys Phe Gln Val Leu Met Lys Asp 50 55 60 gtt ctg aga ccg tacgtg ccc gaa tac aag ggc cac ttg act acc gac 239 Val Leu Arg Pro Tyr ValPro Glu Tyr Lys Gly His Leu Thr Thr Asp 65 70 75 gac gga gac cta tat cttcag cta gaa gac ttg ttg ggt gac ttc act 287 Asp Gly Asp Leu Tyr Leu GlnLeu Glu Asp Leu Leu Gly Asp Phe Thr 80 85 90 95 tcg ccg tgc gtc atg gactgc aag atc ggc gtc agg acg tat ctg gaa 335 Ser Pro Cys Val Met Asp CysLys Ile Gly Val Arg Thr Tyr Leu Glu 100 105 110 gag gaa ctg gcg aaa gccaaa gag aaa ccc aag ttg aga aaa gac atg 383 Glu Glu Leu Ala Lys Ala LysGlu Lys Pro Lys Leu Arg Lys Asp Met 115 120 125 tac gaa aaa atg att cagata gac ccc aac gca cca tcg gag gag gaa 431 Tyr Glu Lys Met Ile Gln IleAsp Pro Asn Ala Pro Ser Glu Glu Glu 130 135 140 cac cga ctg aag ggt gtgaca aaa ccg agg tac atg gtt tgg agg gag 479 His Arg Leu Lys Gly Val ThrLys Pro Arg Tyr Met Val Trp Arg Glu 145 150 155 acg att tcg tcc acg gccacg ttg ggc ttc cgg atc gag ggg atc aag 527 Thr Ile Ser Ser Thr Ala ThrLeu Gly Phe Arg Ile Glu Gly Ile Lys 160 165 170 175 aaa agc gat gga aaatcg agc aag gac ttc aag acg aca aag aac cgg 575 Lys Ser Asp Gly Lys SerSer Lys Asp Phe Lys Thr Thr Lys Asn Arg 180 185 190 gac cag gtg atc gaagcg ttt cga gat ttc gtc gcc ggt ttc ccg cac 623 Asp Gln Val Ile Glu AlaPhe Arg Asp Phe Val Ala Gly Phe Pro His 195 200 205 gta atc ccc aag tacata aac cga ctg aga gcg atc aga gac ata ctg 671 Val Ile Pro Lys Tyr IleAsn Arg Leu Arg Ala Ile Arg Asp Ile Leu 210 215 220 gtg aac tcc aag tttttc act acg cac gag gtg atc ggc agc tcg ctg 719 Val Asn Ser Lys Phe PheThr Thr His Glu Val Ile Gly Ser Ser Leu 225 230 235 ctg ttc gtg cac gacagc aag aac gcc aac ata tgg ctt atc gac ttc 767 Leu Phe Val His Asp SerLys Asn Ala Asn Ile Trp Leu Ile Asp Phe 240 245 250 255 gca aag acg ctcata ctt ccg ccg gac atc cgg atc aac cac acg tcc 815 Ala Lys Thr Leu IleLeu Pro Pro Asp Ile Arg Ile Asn His Thr Ser 260 265 270 gag tgg gtg gtgggc aac cac gag gac ggt tac ctg atc ggt atc aac 863 Glu Trp Val Val GlyAsn His Glu Asp Gly Tyr Leu Ile Gly Ile Asn 275 280 285 aac ctg ctg gacata ttc acc gat atg aac gcc gcc acc gcg ttt ccc 911 Asn Leu Leu Asp IlePhe Thr Asp Met Asn Ala Ala Thr Ala Phe Pro 290 295 300 gtc acg ctc atcgaa gtc acg gcc ccg tcc gaa gtc acc tga 953 Val Thr Leu Ile Glu Val ThrAla Pro Ser Glu Val Thr * 305 310 315 acgccgtcga tccccgccgg taccctgactcgctcggcga cccactcgcc ggtgtcattc 1013 gattccagcc acccactcag tggtcttgcgaatcacgtga cccaccccgt tgacaatgtg 1073 tgataataat aatatgtctg gcgcaaaatattccaaaaag tcttttttaa attacacttt 1133 cgattttcga cgacaaacaa aatgacgacgttttccgtac ctacctactg tagggttcgt 1193 tccgattgca atcataattt attttacccccacccaaccc ccgaaccgtt tatggcccac 1253 cagaggattt gccatcagta ttaaaacaatgatctattat agatgttaaa aaataaatat 1313 tatataatta tacatcatcg cggtgtgttgtgtaatatgc ctattataat atgtactata 1373 ttatacacat agcatattat aaaaatagtatattattata ttatattata ataatattat 1433 ggttatgtgt gtttgtgtgg aaatccaataatataaaata atagttatta tttttaaata 1493 cttgtacgat aatgggacta ctacgtgtgattctcaaatg atatatatat attaatattt 1553 taaacgtaca tttttaattc caaacgtatatgacgtgtgt atatattatt atgatataat 1613 aattactata ctgtgcgtgc gataacataataattttgta cctaatacat caatcaatta 1673 tccactgcag tgtcgtgtgg tttttatttcgttgttttat tttatcgcta tcactaaatt 1733 actattttta ttattattat tttttttttttttcaaaaac tttgttttat aatcagctcc 1793 ctccactacc cttttcacaa cccctcttgtccatgtatta agcaaataat tattttttta 1853 aatacctatc cacgttacaa cgacaataataataacaata atagtaccta tactttattt 1913 ttatttcctc acgaaaacga gaagtcctcatttctttctc ccgttacagt gtgtgtgtgt 1973 gtgtgtgtgt gtgtgtgtgt gtgtgtgtgtgcgtatgtgt atgtgtgaaa tttttgattt 2033 aattatatat tattataatt ttttctccttatatttttat ttattattat aacatttttt 2093 ttgtgtgtac agaatattta aataagacttgtaaaagaaa cccttgttat attattttat 2153 tttttatttc acttcgcaca tgtgtacataataaatcgtt atcgccttaa aaaaaaaaaa 2213 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa aaaaaaa 2270 25 316 PRT Zea mays 25 Thr Arg ProArg Lys Leu Arg Asn Ile Val Gln Trp Thr Pro Phe Phe 1 5 10 15 Gln ThrTyr Lys Lys Gln Arg Tyr Pro Trp Val Gln Leu Ala Gly His 20 25 30 Gln GlyAsn Phe Lys Ala Gly Pro Glu Pro Gly Thr Ile Leu Lys Lys 35 40 45 Leu CysPro Lys Glu Gln Leu Cys Phe Gln Val Leu Met Lys Asp Val 50 55 60 Leu ArgPro Tyr Val Pro Glu Tyr Lys Gly His Leu Thr Thr Asp Asp 65 70 75 80 GlyAsp Leu Tyr Leu Gln Leu Glu Asp Leu Leu Gly Asp Phe Thr Ser 85 90 95 ProCys Val Met Asp Cys Lys Ile Gly Val Arg Thr Tyr Leu Glu Glu 100 105 110Glu Leu Ala Lys Ala Lys Glu Lys Pro Lys Leu Arg Lys Asp Met Tyr 115 120125 Glu Lys Met Ile Gln Ile Asp Pro Asn Ala Pro Ser Glu Glu Glu His 130135 140 Arg Leu Lys Gly Val Thr Lys Pro Arg Tyr Met Val Trp Arg Glu Thr145 150 155 160 Ile Ser Ser Thr Ala Thr Leu Gly Phe Arg Ile Glu Gly IleLys Lys 165 170 175 Ser Asp Gly Lys Ser Ser Lys Asp Phe Lys Thr Thr LysAsn Arg Asp 180 185 190 Gln Val Ile Glu Ala Phe Arg Asp Phe Val Ala GlyPhe Pro His Val 195 200 205 Ile Pro Lys Tyr Ile Asn Arg Leu Arg Ala IleArg Asp Ile Leu Val 210 215 220 Asn Ser Lys Phe Phe Thr Thr His Glu ValIle Gly Ser Ser Leu Leu 225 230 235 240 Phe Val His Asp Ser Lys Asn AlaAsn Ile Trp Leu Ile Asp Phe Ala 245 250 255 Lys Thr Leu Ile Leu Pro ProAsp Ile Arg Ile Asn His Thr Ser Glu 260 265 270 Trp Val Val Gly Asn HisGlu Asp Gly Tyr Leu Ile Gly Ile Asn Asn 275 280 285 Leu Leu Asp Ile PheThr Asp Met Asn Ala Ala Thr Ala Phe Pro Val 290 295 300 Thr Leu Ile GluVal Thr Ala Pro Ser Glu Val Thr 305 310 315 26 25 DNA ArtificialSequence Primer 26 accgcttcca ccatcgccac tcgtc 25 27 30 DNA ArtificialSequence Primer 27 ccttagacgc agcctttggg tccttcttaa 30 28 36 DNAArtificial Sequence Primer 28 tcgacccacg cgtccgaaaa aaaaaaaaaa aaaaaa 3629 21 PRT Artificial Sequence Consensus Sequence 29 Ile Leu Leu Glu AsnLeu Thr Ser Arg Tyr Glu Val Pro Cys Val Leu 1 5 10 15 Asp Leu Lys MetGly 20 30 33 PRT Artificial Sequence Consensus Sequence 30 Leu Lys XaaPro Glu His Gln Val Ala Gly His Xaa Ala Xaa Xaa Gly 1 5 10 15 Lys XaaGly Pro Leu Val Asp Asp Xaa Gly Xaa Phe Tyr Lys Pro Leu 20 25 30 Gln 3133 PRT Artificial Sequence Consensus Sequence 31 Leu Lys Xaa Pro Glu HisGln Val Ala Gly His Xaa Ala Xaa Xaa Gly 1 5 10 15 Lys Xaa Gly Pro LeuIle Asp Asp Xaa Gly Xaa Phe Tyr Lys Pro Leu 20 25 30 Gln 32 33 PRTArtificial Sequence Consensus Sequence 32 Leu Lys Xaa Pro Asp His GlnVal Ala Gly His Xaa Ala Xaa Xaa Gly 1 5 10 15 Lys Xaa Gly Pro Leu ValAsp Asp Xaa Gly Xaa Phe Tyr Lys Pro Leu 20 25 30 Gln 33 33 PRTArtificial Sequence Consensus Sequence 33 Leu Lys Xaa Pro Asp His GlnVal Ala Gly His Xaa Ala Xaa Xaa Gly 1 5 10 15 Lys Xaa Gly Pro Leu IleAsp Asp Xaa Gly Xaa Phe Tyr Lys Pro Leu 20 25 30 Gln 34 41 PRTArtificial Sequence Consensus Sequence 34 Val Leu Xaa Asp Leu Xaa XaaXaa Xaa Xaa Xaa Pro Ser Val Met Asp 1 5 10 15 Val Lys Xaa Gly Ser ArgThr Trp Xaa Xaa Xaa Xaa Xaa Glu Xaa Tyr 20 25 30 Ile Xaa Lys Cys Leu XaaLys Asp Arg 35 40 35 41 PRT Artificial Sequence Consensus Sequence 35Val Leu Xaa Asp Leu Xaa Xaa Xaa Xaa Xaa Xaa Pro Ser Val Met Asp 1 5 1015 Ile Lys Xaa Gly Ser Arg Thr Trp Xaa Xaa Xaa Xaa Xaa Glu Xaa Tyr 20 2530 Ile Xaa Lys Cys Leu Xaa Lys Asp Arg 35 40 36 41 PRT ArtificialSequence Consensus Sequence 36 Val Leu Xaa Asp Leu Xaa Xaa Xaa Xaa XaaXaa Pro Cys Val Met Asp 1 5 10 15 Val Lys Xaa Gly Ser Arg Thr Trp XaaXaa Xaa Xaa Xaa Glu Xaa Tyr 20 25 30 Ile Xaa Lys Cys Leu Xaa Lys Asp Arg35 40 37 41 PRT Artificial Sequence Consensus Sequence 37 Val Leu XaaAsp Leu Xaa Xaa Xaa Xaa Xaa Xaa Pro Cys Val Met Asp 1 5 10 15 Ile LysXaa Gly Ser Arg Thr Trp Xaa Xaa Xaa Xaa Xaa Glu Xaa Tyr 20 25 30 Ile XaaLys Cys Leu Xaa Lys Asp Arg 35 40

1. An isolated nucleic acid comprising a member selected from the groupconsisting of: (a) a polynucleotide having at least 75% sequenceidentity compared to the full-length of the sequence of SEQ ID NOS:1, 3,5, 7, 9, 11, 13, 15, 17-20, 22, or 24; wherein the % sequence identityis determined by GAP 10 analysis using default parameters; (b) apolynucleotide which encodes a polypeptide of SEQ ID NOS:2, 4, 6, 8, 10,12, 14, 16, 21, 23, 25, or 29-37; (c) a polynucleotide amplified from aplant nucleic acid library using the primers of SEQ ID NOS: 26 and 27,or primers determined by using Vector NTI Suite, InforMax Version 5; (d)a polynucleotide comprising at least 20 contiguous bases of SEQ IDNOS:1, 3, 5, 7, 9, 11, 13, 15, 17-20, 22,or24; (e) a polynucleotidecomprising at least 25 nucleotides in length which hybridizes, underhigh stringency conditions and a wash in 0.1×SSC at 60° C., to apolynucleotide having the sequence set forth in SEQ ID NOS:1, 3, 5, 7,9, 11, 13, 15, 17-20, 22, or 24; (f) a polynucleotide coding for a plantinositol polyphosphate kinase (IPPK) protein other than fromArabidopsis; (g) a polynucleotide having the sequence set forth in SEQID NOS:1, 3, 5, 7, 9, 11, 13, 15, 17-20, 22, or 24; and (h) apolynucleotide complementary to a polynucleotide of (a) through (g). 2.The isolated nucleic acid of claim 1, wherein the polynucleotide is froma monocot or dicot.
 3. A vector comprising at least one nucleic acid ofclaim
 1. 4. An expression cassette comprising at least one nucleic acidof claim 1 operably linked to a promoter, wherein the nucleic acid is insense or antisense orientation.
 5. The expression cassette of claim 4,wherein the nucleic acid is operably linked in antisense orientation tothe promoter.
 6. A non-human host cell containing at least oneexpression cassette of claim
 4. 7. The host cell of claim 6 that is aplant cell.
 8. A transgenic plant comprising at least one expressioncassette of claim
 4. 9. The transgenic plant of claim 8, wherein theplant is corn, soybean, sorghum, wheat, rice, alfalfa, safflower,sunflower, canola, cotton, or turf grass.
 10. A seed from the transgenicplant of claim
 8. 11. The seed from the transgenic plant of claim
 9. 12.An isolated protein comprising a member selected from the groupconsisting of: (a) a polypeptide comprising at least 25 contiguous aminoacids of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 21, 23, or 25; (c) apolypeptide comprising at least 60% sequence identity compared to thefull-length of SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 16, 21, 23, or 25;wherein the percent sequence identity is based on the entire sequenceand is determined by GAP 10 analysis using default parameters; (d) apolypeptide encoded by a nucleic acid of claim 1; (e) a polypeptideencoded by a nucleic acid of SEQ ID NOS:1, 3, 5, 7, 9, 11, 13, or 15;(f) a polypeptide encoded by a nucleic acid of SEQ ID NOS: 20, 22, or24; and (g) a polypeptide having the sequence set forth in SEQ ID NOS:2, 4, 6, 8, 10, 12, 14, 16, 21, 23, or
 25. 13. An isolated ribonucleicacid sequence encoding a protein of claim
 12. 14. A method formodulating inositol polyphosphate kinase (IPPK) activity or levels in ahost cell, comprising: (a) transforming a host cell with at least oneexpression cassette of claim 4; and (b) growing the transformed hostcell under conditions sufficient to modulate IPPK activity in the hostcell.
 15. The method of claim 14, wherein the host cell is a plant cell.16. The method of claim 15, wherein the plant cell is from a monocot ora dicot.
 17. A plant produced by the method of claim
 14. 18. Thetransgenic plant of claim 17, wherein the plant is corn, soybean,sorghum, wheat, rice, alfalfa, safflower, sunflower, canola, cotton, orturf grass.
 19. The method of claim 15 wherein the level of phytate isreduced.
 20. The method of claim 15 wherein the level of non-phytatephosphorous is increased.
 21. A method of decreasing the level ofphosphorous in non-ruminant animal waste comprising providing saidanimal feed from a plant produced by the method of claim
 14. 22. Amethod of improving the nutritional value of animal feed, comprising:(a) transforming a plant host cell with at least one expression cassetteof claim 4; and (b) growing the transformed host cell under conditionssufficient to modulate IPPK activity in the host cell; (c) generating aplant with the transformed genotype; and (d) producing animal feed fromthe plant, wherein the animal feed has improved the nutritional value.23. The method of claim 22, wherein the plant cell is from a monocot ora dicot.
 24. A plant produced by the method of claim
 22. 25. A seed froma plant of claim
 24. 26. The transgenic plant of claim 24, wherein theplant is corn, soybean, sorghum, wheat, rice, safflower, sunflower, orcanola.
 27. The method of claim 22, wherein the level of phytate isreduced.
 28. The method of claim 22, wherein the level of non-phytatephosphorous is increased.
 29. A method of decreasing the level ofphosphorous in non-ruminant animal waste comprising providing saidanimal feed from a plant produced by the method of claim
 22. 30. Anisolated protein containing a polypeptide sequence selected from thegroup consisting of SEQ ID NOS: 30-33.
 31. An isolated proteincontaining the polypeptide sequence selected from the group consistingof SEQ ID NOS: 34-37.
 32. A method of increasing the level of availablephosphorous in animal feed, comprising: (a) transforming a plant hostcell with at least one expression cassette of claim 4; and (b) growingthe transformed host cell under conditions sufficient to modulate IPPKactivity in the host cell; (c) generating a plant with the transformedgenotype; and (d) producing animal feed from the plant, wherein theanimal feed has an increased level of available phosphorous.
 33. Themethod of claim 32, wherein the plant cell is from a monocot or a dicot.34. A plant produced by the method of claim
 32. 35. A seed from a plantof claim
 34. 36. The transgenic plant of claim 34, wherein the plant iscorn, soybean, sorghum, wheat, rice, safflower, sunflower, or canola.37. The method of claim 32, wherein the level of phytate is reduced. 38.A method of decreasing the level of phosphorous in non-ruminant animalwaste comprising providing said animal feed from a plant produced by themethod of claim
 32. 39. A method of altering plant phenotype comprising:(a) transforming a plant host cell with at least one IPPK polynucleotideof claim 1 and at least one polynucleotide of interest; (b) growing thetransformed host cell under conditions sufficient to modulate theactivity of IPPK and the polynucleotide of interest in the host cell;and (c) generating a plant with an altered phenotype.
 40. The method ofclaim 39, wherein the activity of IPPK is downregulated while theactivity of the polynucleotide of interest is up-regulated.
 41. Themethod of claim 40, wherein the polynucleotide of interest ismyo-inositol monophosphatase (IMP) or phytase.
 42. The method of claim39, wherein the activity of IPPK and the activity of the polynucleotideof interest are downregulated.
 43. The method of claim 42, wherein thepolynucleotide of interest is inositol 1,3,4-trisphosphate ⅚-kinase(ITPK) or myo-inositol 1-phosphate synthase (MI1PS).
 44. A transgenicplant produced by the method of claim
 39. 45. The transgenic plant ofclaim 44, wherein the plant is corn, soybean, sorghum, wheat, rice,alfalfa, safflower, sunflower, canola, cotton, or millet.
 46. A seedfrom a plant of claim 44.